Inhibitors of macrophage migration inhibitory factor and methods for identifying the same

ABSTRACT

Inhibitors of MIF are provided which have utility in the treatment of a variety of disorders, including the treatment of pathological conditions associated with MIF activity. The inhibitors of MIF have the following structures: 
                         
including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein n, R 1 , R 2 , R 3 , R 4 , X, Y and Z are as defined herein. Compositions containing an inhibitor of MIF in combination with a pharmaceutically acceptable carrier are also provided, as well as methods for use of the same.

RELATED APPLICATION

This application is a division of U.S. application Ser. No. 10/156,650,filed May 24, 2002, which claims the benefit of U.S. ProvisionalApplication No. 60/293,642, filed May 24, 2001. All above-referencedprior applications are incorporated by reference herein in theirentirety and are hereby made a portion of this specification.

FIELD OF THE INVENTION

This invention relates generally to inhibitors of macrophage migrationinhibitory factor (MIF), methods for identifying MIF inhibitors, and tomethods of treating MIF-related disorders by administration of suchinhibitors.

BACKGROUND OF THE INVENTION

The lymphokine, macrophage migration inhibitory factor (MIF), has beenidentified as a mediator of the function of macrophages in host defenseand its expression correlates with delayed hypersensitivity,immunoregulation, inflammation, and cellular immunity. See Metz andBucala, Adv. Immunol. 66:197–223, 1997. Macrophage migration inhibitoryfactors (MIFs), which are between 12–13 kilodaltons (kDa) in size, havebeen identified in several mammalian and avian species; see, forexample, Galat et al., Fed. Eur. Biochem. Soc. 319:233–236, 1993; Wistowet al., Proc. Natl. Acad. Sci. USA 90:1272–1275, 1993; Weiser et al.,Proc. Natl. Acad. Sci. USA 86:7522–7526, 1989; Bernhagen et al., Nature365:756–759, 1993; Blocki et al., Protein Science 2:2095–2102, 1993; andBlocki et al., Nature 360:269–270, 1992. Although MIF was firstcharacterized as being able to block macrophage migration, MIF alsoappears to effect macrophage adherence; induce macrophage to expressinterleukin-1-beta, interleukin-6, and tumor necrosis factor alpha;up-regulate HLA-DR; increase nitric oxide synthase and nitric oxideconcentrations; and activate macrophage to kill Leishmania donovanitumor cells and inhibit Mycoplasma avium growth, by a mechanismdifferent from that effected by interferon-gamma. In addition to itspotential role as an immunoevasive molecule, MIF can act as animmunoadjuvant when given with bovine serum albumin or HIV gp120 inincomplete Freunds or liposomes, eliciting antigen induced proliferationcomparable to that of complete Freunds. Also, MIF has been described asa glucocorticoid counter regulator and angiogenic factor. As one of thefew proteins that is induced and not inhibited by glucocorticoids, itserves to attenuate the immunosuppressive effects of glucocorticoids. Assuch, it is viewed as a powerful element that regulates theimmunosuppressive effects of glucocorticoids. Hence, when itsactivities/gene expression are overinduced by the administration ofexcess exogenous glucocorticoids (for example when clinical indicated tosuppress inflammation, immunity and the like), there is significanttoxicity because MIF itself exacerbates the inflammatory/immuneresponse. See Buccala et al., Ann. Rep. Med. Chem. 33:243–252, 1998.

While MIF is also thought to act on cells through a specific receptorthat in turn activates an intracellular cascade that includes erkphosphorylation and MAP kinase and upregulation of matrixmetalloproteases, c-jun, c-fos, and IL-1 mRNA (see Onodera et al., J.Biol. Chem. 275:444–450, 2000), it also possesses endogenous enzymeactivity as exemplified by its ability to tautomerize the appropriatesubstrates (e.g., dopachrome). Further, it remains unclear whether thisenzymatic activity mediates the biological response to MIF and theactivities of this protein in vitro and in vivo. While site directedmutagenesis of MIF has generated mutants which possess full intrinsicactivity, yet fail to possess enzyme activity (Hermanowski-Vosatka etal., Biochemistry 38:12841–12849, 1999), Swope et al. have described adirect link between cytokine activity and the catalytic site for MIF(Swope et al., EMBO J. 7(13):3534–3541, 1998). Accordingly, it isunclear that strategies to identify inhibitors of MIF activity throughinhibition of dopachrome tautomerase alone yields inhibitors of MIFactivity of clinical value. The ability to evaluate the inhibition ofMIF to its cell surface receptor is also limited since no high affinityreceptor is currently known.

The interest in developing MIF inhibitors derives from the observationthat MIF is known for its cytokine activity concentrating macrophages atsites of infection, and cell-mediated immunity. Moreover, MIF is knownas a mediator of macrophage adherence, phagocytosis, and tumoricidalactivity. See Weiser et al., J. Immunol. 147:2006–2011, 1991. Hence, theinhibition of MIF results in the indirect inhibition of cytokines,growth factors, chemokines and lymphokines that the macrophage mayotherwise bring to a site of inflammation. Human MIF cDNA has beenisolated from a T-cell line, and encodes a protein having a molecularmass of about 12.4 kDa with 115 amino acid residues that form ahomotrimer as the active form (Weiser et al., Proc. Natl. Acad. Sci. USA86:7522–7526, 1989). While MIF was originally observed in activatedT-cells, it has now been reported in a variety of tissues including theliver, lung, eye lens, ovary, brain, heart, spleen, kidney, muscle, andothers. See Takahashi et al., Microbiol. Immunol. 43(1):61–67, 1999.Another characteristic of MIF is its lack of a traditional leadersequence (i.e., a leaderless protein) to direct classical secretionthrough the ER/Golgi pathway.

A MIF inhibitor (and a method to identify MIF inhibitors) that act byneutralizing the cytokine activity of MIF presents significantadvantages over other types of inhibitors. For example, the link betweentautomerase activity alone and the inflammatory response iscontroversial. Furthermore, inhibitors that act intracellularly areoften toxic by virtue of their action on related targets or theactivities of the target inside cells. Small molecule inhibitors of theligand receptor complex are difficult to identify let alone optimize anddevelop. The ideal inhibitor of a cytokine like MIF is one that altersMIF itself so that when released from the cell it is effectivelyneutralized. A small molecule with this activity is superior toantibodies because of the fundamental difference between proteins andchemicals as drugs.

SUMMARY OF THE INVENTION

As MIF has been identified in a variety of tissues and has beenassociated with numerous pathological events, there exists a need in theart to identify inhibitors of MIF. There is also a need pharmaceuticalcompositions containing such inhibitors, as well as methods relating tothe use thereof to treat, for example, immune related disorders or otherMIF induced pathological events, such as tumor associated angiogenesis.The preferred embodiments may fulfill these needs, and provided otheradvantages as well.

In preferred embodiments, inhibitors of MIF are provided that have thefollowing general structures (Ia) and (Ib):

including stereoisomers, prodrugs, and pharmaceutically acceptable saltsthereof, wherein n, R₁, R₂, R₃, R₄, X, Y Z are as defined below.

The MIF inhibitors of preferred embodiments have utility over a widerange of therapeutic applications, and may be employed to treat avariety of disorders, illnesses, or pathological conditions including,but not limited to, a variety of immune related responses, tumor growth(e.g., prostate cancer, etc.), glomerulonephritis, inflammation,malarial anemia, septic shock, tumor associated angiogenesis,vitreoretinopathy, psoriasis, graft versus host disease (tissuerejection), atopic dermatitis, rheumatoid arthritis, inflammatory boweldisease, otitis media, Crohn's disease, acute respiratory distresssyndrome, delayed-type hypersensitivity, and others. See, e.g., Metz andBucala (supra); Swope and Lolis, Rev. Physiol. Biochem. Pharmacol139:1–32, 1999; Waeber et al., Diabetes M. Res. Rev. 15(1):47–54, 1999;Nishihira, Int. J. Mol. Med. 2(1):17–28, 1998; Bucala, Ann. N.Y. Acad.Sci. 840:74–82, 1998; Bernhagen et al., J. Mol. Med. 76(3–4):151–161,1998; Donnelly and Bucala, Mol. Med. Today 3(11):502–507, 1997; Bucalaet al., FASEB J. 10(14):1607–1613, 1996. Such methods includeadministering an effective amount of one or more inhibitors of MIF asprovided by the preferred embodiments, preferably in the form of apharmaceutical composition, to an animal in need thereof. Accordingly,in another embodiment, pharmaceutical compositions are providedcontaining one or more inhibitors of MIF of preferred embodiments incombination with a pharmaceutically acceptable carrier and/or diluent.

One strategy of a preferred embodiment characterizes molecules thatinteract with MIF so as to induce a conformational change in MIF and assuch a loss of immunoreactivity to a monoclonal antibody. This change,when identified by screening, identifies small molecule inhibitors ofMIF. This particular aspect may be extended to any bioactive polypeptidewhere loss of immunoreactivity may act as a surrogate for activity(e.g., cytokine activity, enzymatic activity, co-factor activity, or thelike).

In a first embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ is selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; with the provisos that R₄ is not hydrogen or methylwhen R₁ is phenyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is—C(═O)OCH₂CH₃; R₄ is not methyl when R₁ is methyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —NO₂; R₄ is not —CH₂CH₂OH when R₁ ishydrogen or methyl, R₂ is 7-chloro, R₃ is hydrogen, X is oxygen and Y is—C(═O)OCH₂CH₃; and R₄ is not methyl when R₁ is methyl, R₂ is hydrogen or7-chloro, R₃ is hydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃.

In a second embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂—; m is 0, 1, or 2; n is 1; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁is methyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is —NO₂; R₄is not —CH₂CH₂OH when R₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ ishydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃; and R₄ is not methyl whenR₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen, X is oxygen,and Y is —C(═O)OCH₂CH₃.

In aspects of the second embodiment, X is oxygen; or Y is —C(═O)OCH₂CH₃;or Y is —NO₂; or R₄ is

In a third embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y is —NO₂; Z is —CH₂— or—C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the proviso that when nis 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisothat R₄ is not methyl when R₁ is methyl, R₂ and R₃ are both hydrogen, Xis oxygen, and Y is —NO₂.

In aspects of the third embodiment, X is oxygen; or Z is —CH₂— and n is1; or R₄ is

In a fourth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y is —C(═O)OCH₂CH₃; Z is —CH₂—or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the proviso that whenn is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, and X is oxygen; R₄ is not —CH₂CH₂OH when R₁ is hydrogen ormethyl, R₂ is 7-chloro, R₃ is hydrogen, and X is oxygen; and R₄ is notmethyl when R₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen,and X is oxygen.

In aspects of the fourth embodiment, X is oxygen; or Z is —CH₂— and n is1; or R₄ is

In a fifth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is

and R₅ and R₆ independently include hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle.

In aspects of the fifth embodiment, X is oxygen; or Z is —CH₂— and n is1; or Y is —C(═O)OCH₂CH₃; or Y is —NO₂.

In a sixth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is

and R₅ and R₆ independently include hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle.

In aspects of the sixth embodiment, X is oxygen; or Z is —CH₂— and n is1; or Y is —C(═O)OCH₂CH₃; or Y is —NO₂.

In a seventh embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen; Y includes —NO, —NO₂, —C(═O)R₅, —C(═O)OR₅,—(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is —CH₂— or—C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the proviso that when nis 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁ is methyl, R₂and R₃ are both hydrogen, and Y is —NO₂; R₄ is not —CH₂CH₂OH when R₁ ishydrogen or methyl, R₂ is 7-chloro, R₃ is hydrogen, and Y is—C(═O)OCH₂CH₃; and R₄ is not methyl when R₁ is methyl, R₂ is hydrogen or7-chloro, R₃ is hydrogen, and Y is —C(═O)OCH₂CH₃.

In aspects of the seventh embodiment, Z is —CH₂— and n is 1; or Y is—C(═O)OCH₂CH₃; or Y is —NO₂; or R₄ is

In an eighth embodiment, a composition is provided including a compoundof the first embodiment in combination with a pharmaceuticallyacceptable carrier or diluent.

In a ninth embodiment, a method for reducing MIF activity in a patientin need thereof is provided, including administering to the patient aneffective amount of a compound having the structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁is methyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is —NO₂; R₄is not —CH₂CH₂OH when R₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ ishydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃; and R₄ is not methyl whenR₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen, X is oxygen,and Y is —C(═O)OCH₂CH₃.

In a tenth embodiment, a method is provided for treating inflammation ina warm-blooded animal, including administering to the animal aneffective amount of the compound of the first embodiment.

In an eleventh embodiment, a method is provided for treating septicshock in a warm-blooded animal, including administering to the animal aneffective amount of the compound of the first embodiment.

In a twelfth embodiment, a method is provided for treating arthritis ina warm-blooded animal, including administering to the animal aneffective amount of the compound of the first embodiment.

In a thirteenth embodiment, a method is provided for treating cancer ina warm-blooded animal, including administering to the animal aneffective amount of the compound of the first embodiment.

In a fourteenth embodiment, a method is provided for treating acuterespiratory distress syndrome in a warm-blooded animal, includingadministering to the animal an effective amount of the compound of thefirst embodiment.

In a fifteenth embodiment, a method is provided for treating aninflammatory disease in a warm-blooded animal, including administeringto the animal an effective amount of the compound of the firstembodiment. In aspects of the fifteenth embodiment, the inflammatorydisease may include rheumatoid arthritis, osteoarthritis, inflammatorybowel disease, and asthma.

In a sixteenth embodiment, a method is provided for treating anautoimmune disorder in a warm-blooded animal, including administering tothe animal an effective amount of the compound of the first embodiment.In aspects of the sixteenth embodiment, the autoimmune disorder includesdiabetes, asthma, and multiple sclerosis.

In a seventeenth embodiment, a method is provided for suppressing animmune response in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the first embodiment.

In an eighteenth embodiment, a method is provided for decreasingangiogenesis in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the first embodiment.

In a nineteenth embodiment, a method is provided for treating a diseaseassociated with excess glucocorticoid levels in a warm-blooded animal,including administering to the animal an effective amount of thecompound of the first embodiment. In an aspect of the nineteenthembodiment, the disease is Cushing's disease.

In an twentieth embodiment, a method is provided for detecting an agentthat modulates MIF activity, including contacting a sample containingMIF with an agent; and detecting the ability of the agent to modulateMIF by determining a differential ability of an antibody to bind MIF. Inan aspect of the twentieth embodiment, the antibody is a monoclonalantibody. In an aspect of the twentieth embodiment, MIF includes fusionproteins, mutants or variants thereof.

In a twenty first embodiment, a method is provided for using antibodybinding as a surrogate marker for screening for an agent that modulatesthe activity of a polypeptide, including contacting the polypeptide witha suspected modulating agent, contacting the polypeptide with amonoclonal antibody, and detecting a differential activity of thepolypeptide relative to a control.

In a twenty second embodiment, a compound is provided having astructure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat: R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁is methyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is —NO₂; R₄is not —CH₂CH₂OH when R₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ ishydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃; and R₄ is not methyl whenR₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen, X is oxygen,and Y is —C(═O)OCH₂CH₃.

In a twenty third embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂—; m is 0, 1, or 2; n is 1; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁is methyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is —NO₂; R₄is not —CH₂CH₂OH when R₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ ishydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃; and R₄ is not methyl whenR₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen, X is oxygen,and Y is —C(═O)OCH₂CH₃.

In aspects of the twenty third embodiment, R₁ is —NCH₂CH₂CH₂N(CH₃)₂; orX is oxygen; or Y is —C(═O)OCH₂CH₃; or Y is —NO₂; or R₄ is

In a twenty fourth embodiment, a compound is provided having astructure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y is —C(═O)OCH₂CH₃; Z is —CH₂—or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the proviso that whenn is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆;R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, and X is oxygen; R₄ is not —CH₂CH₂OH when R₁ is hydrogen ormethyl, R₂ is 7-chloro, R₃ is hydrogen, and X is oxygen; and R₄ is notmethyl when R₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen,and X is oxygen.

In aspects of the twenty forth embodiment, X is oxygen; or Z is —CH₂—and n is 1; or R₄ is

In a twenty fifth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ is —NCH₂CH₂CH₂N(CH₃)₂; R₂ and R₃independently include halogen, —R₅, —OR₅, —SR₅, and —NR₅R₆; R₄ includes—CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅ and R₆independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl.

In aspects of the twenty fifth embodiment, Z is —CH₂— and n is 1; or Yis —C(═O)OCH₂CH₃; or Y is —NO₂; or R₄ is

In a twenty sixth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y is —NO₂; Z is —CH₂— or—C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the proviso that when nis 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisothat R₄ is not methyl when R₁ is methyl, R₂ and R₃ are both hydrogen,and X is oxygen.

In aspects of the twenty sixth embodiment, R₁ is —NCH₂CH₂CH₂N(CH₃)₂; orX is oxygen; or Z is —CH₂— and n is 1; or R₄ is

In a twenty seventh embodiment, a compound is provided having astructure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes

R₅ and R₆ independently include hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl.

In aspects of the twenty seventh embodiment, R₁ is —NCH₂CH₂CH₂N(CH₃)₂;or X is oxygen; or Z is —CH₂— and n is 1; or Y is —C(═O)OCH₂CH₃; or Y is—NO₂.

In a twenty eighth embodiment, a method is provided for reducing MIFactivity in a patient in need thereof, including administering to thepatient an effective amount of a compound having the structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Y includes —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, and —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1, or 2, with the provisothat when n is 0, Z is —C(═O)—; R₁ includes hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ independently include hydrogen, alkyl, substitutedalkyl, aryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, anddialkyl; R₂ and R₃ independently include halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ includes —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅and R₆ independently include hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl; or R₅ and R₆ taken together with a nitrogen atom towhich they are attached form a heterocycle or substituted heterocycle;R₇ includes alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ includeshydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; with the provisosthat R₄ is not hydrogen or methyl when R₁ is phenyl, R₂ and R₃ are bothhydrogen, X is oxygen, and Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁is methyl, R₂ and R₃ are both hydrogen, X is oxygen, and Y is —NO₂; R₄is not —CH₂CH₂OH when R₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ ishydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃; and R₄ is not methyl whenR₁ is methyl, R₂ is hydrogen or 7-chloro, R₃ is hydrogen, X is oxygen,and Y is —C(═O)OCH₂CH₃.

In a twenty ninth embodiment, a method is provided for treatinginflammation in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the twenty eighthembodiment.

In a thirtieth embodiment, a method is provided for treating septicshock in a warm-blooded animal, including administering to the animal aneffective amount of the compound of the twenty eighth embodiment.

In a thirty first embodiment, a method is provided for treatingarthritis in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the twenty eighthembodiment.

In a thirty second embodiment, a method is provided for treating cancerin a warm-blooded animal, including administering to the animal aneffective amount of the compound of the twenty eighth embodiment.

In a thirty third embodiment, a method is provided for treating acuterespiratory distress syndrome in a warm-blooded animal, includingadministering to the animal an effective amount of the compound of thetwenty eighth embodiment.

In a thirty fourth embodiment, a method is provided for treating aninflammatory disease in a warm-blooded animal, including administeringto the animal an effective amount of the compound of the twenty eighthembodiment. In aspects of the thirty fourth embodiment, the inflammatorydisease includes rheumatoid arthritis, osteoarthritis, inflammatorybowel disease, and asthma.

In a thirty fifth embodiment, a method is provided for treating anautoimmune disorder in a warm-blooded animal, including administering tothe animal an effective amount of the compound of the twenty eighthembodiment. In aspects of the thirty fifth embodiment, the autoimmunedisorder includes diabetes, asthma, and multiple sclerosis.

In a thirty sixth embodiment, a method is provided for suppressing animmune response in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the twenty eighthembodiment.

In a thirty seventh embodiment, a method is provided for decreasingangiogenesis in a warm-blooded animal, including administering to theanimal an effective amount of the compound of the twenty eighthembodiment.

In a thirty eighth embodiment, a method is provided for treating adisease associated with excess glucocorticoid levels in a warm-bloodedanimal, including administering to the animal an effective amount of thecompound of the twenty eighth embodiment. In an aspect of the thirtyeighth embodiment, the disease is Cushing's disease.

In a thirty ninth embodiment, a process is provided for preparing acompound including the steps of reacting a compound of Formula I:

with a compound of Formula II:

thereby obtaining a compound of Formula III:

wherein R₃ includes R₄ is amino, substituted amino hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, andsubstituted heterocyclealkyl; and reacting the compound of Formula IIIwith a compound including X—R₄, wherein X includes Cl, Br, and I, andwherein R₄ includes hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and aminoalkyl R′R″N(CH₂)_(x)—, wherein R′and R″ independently include hydrogen, alkyl, substituted alkyl, aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, and dialkyl, wherein xis 2 to 4, thereby obtaining a compound of Formula IV:

wherein the compound of Formula IV is suitable for use as a MIFinhibitor.

In aspects of the thirty ninth embodiment, R₄ is

In a fortieth embodiment, a process is provided for preparing a compoundincluding the steps of reacting a compound of Formula AI:

with a compound of Formula II:

thereby obtaining a compound of Formula AIII:

wherein R₃ includes R₄ is amino, substituted amino hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, andsubstituted heterocyclealkyl; and reacting the compound of Formula AIIIwith a compound including X—R₄, wherein X includes Cl, Br, and I, andwherein R₄ includes hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, and aminoalkyl R′R″N(CH₂)_(x)—, wherein R′and R″ independently include hydrogen, alkyl, substituted alkyl, aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, and dialkyl, wherein xis 2 to 4, thereby obtaining a compound of Formula AIV:

wherein the compound of Formula IV is suitable for use as a MIFinhibitor.

In aspects of the fortieth embodiment, R₄ is

In a forty first embodiment, a pharmaceutical composition is providedfor treating a disease or disorder wherein MIF is pathogenic, thepharmaceutical composition including a MIF inhibiting compound and adrug for treating the disease or disorder, wherein the drug has nomeasurable MIF inhibiting activity.

In a forty second embodiment, a pharmaceutical composition is providedfor treating a disease or disorder wherein MIF is pathogenic, thepharmaceutical composition including a MIF inhibiting compound and adrug selected from the group consisting of nonsteroidalanti-inflammatory drugs, anti-infective drugs, beta stimulants,steroids, antihistamines, anticancer drugs, asthma drugs, sepsis drugs,arthritis drugs, and immunosuppresive drugs.

These and other embodiments and aspects thereof will be apparent uponreference to the following detailed description. To this end, variousreferences are set forth herein which describe in more detail certainprocedures, compounds and/or compositions, and are hereby incorporatedby reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1B are scanned images of an autoradiogram demonstratingantibody immunoprecipitation with an anti-MIF monoclonal antibody (FIG.1A) and anti-MIF polyclonal sera (FIG. 1B) in cytosolic extracts (C) aswell as conditioned media (M) of THP-1 cells following LPS stimulationand treatment with various micromolar concentrations of compound 7e.Also depicted are the results of tautomerase activity detected in thevarious fractions. Plus signs indicate tautomerase activity, minus signsindicate no detectable activity, and +/−signs indicating partialactivity.

FIG. 2 is a graph depicting enzyme linked immunosorbant assay (ELISA)results following treatment of LPS stimulated THP-1 cells with fiveanalogs of the presently claimed composition. The ability of each analogto inhibit monoclonal antibody binding is depicted and is dosedependent.

FIG. 3 is a graphical representation of the immunoreactivity of MIF inconditioned medium using ELISA following stimulation of THP-1 cells withLPS and addition of 10 μM compound 7e at various times during culture.In this Figure, LPS was added at time point zero, while compound 7e wasadded at 0, 2, 4, and 6 hours following LPS treatment. Six groups ofTHP1 cells were employed in this experiment, all cultured under standardmedia conditions. At the initiation of the experiment, buffer only wasadded to Group 1 cells and buffer containing compound 7e was added toGroup 2 cells. Compound 7e in buffer was added to each other group atvarious times thereafter, Group 3 at 2 hrs, Group 4 at 4 hrs, Group 5 at6 hrs and Group 6 at 22 hrs. Samples were taken from each group at theindicated times after buffer or buffer plus sample was added and assayedfor detectable levels of MIF using the anti-MIF monoclonal antibody. Inthe absence of compound (Group 1) the level of detectable MIF increasedthroughout the time course of the experiment. In the presence ofcompound, detection of MIF is blocked.

FIG. 4 is a graphical representation of an ELISA-based experiment,following the effect of compound 7e on MIF detection. In thisexperimental design, the test sample is pre-conditioned cell culturemedia, clarified of cellular debris. The starting concentration of MIFin the test sample was calculated to be approximately 22 ng/ml. Compoundis added at varying times after beginning incubation at 37° C. Eachsample is then incubated for an additional 30 minutes before thedetectable level of MIF is again determined.

FIG. 5 is a bar graph representing the relative percent of MIF presentin conditioned media from compound 7e treated and LPS induced RAW 264.7cells compared to a control cell population that was not treated withthe compound as measured by ELISA. The top panel demonstrates Westernblots of the same fractions as measured by ELISA in the bottom panel.

FIG. 6 is a bar graph representing the percent of MIF present inconditioned media from compound 7e treated and TSST-1 induced RAW 264.7cells compared to a control cell population that was not treated by thecompound as measured by ELISA. The top panel demonstrates Western blotsof the same fractions measured by ELISA in the bottom panel.

FIGS. 7A–7B are graphical representations of HPLC detection of compound7e in mouse serum following intraperitoneal injection of compound 7e(FIG. 7A) or oral gavage administration of 20 mg of compound 7e (FIG.7B). Results are depicted as a Mean+/−SEM (N=5 mice).

FIG. 8 is a graphical representation of ELISA detected MIF release inmouse serum at various times following LPS/galactosamine challenge.Results are presented as Mean+/−SEM (N=5).

FIGS. 9A–9B graphically illustrate ELISA data of serum MIFconcentrations in ng/ml five hours following a 10 mg/kg LPS challenge(FIG. 9A) or normalized serum MIF four hours following a 5 mg/kg LPSchallenge (FIG. 9B) in the presence or absence of compound 7e (0.4 mg/20gram mouse).

FIG. 10 is a graphical representation of ELISA measurementsdemonstrating the correlation between serum IL-1β levels in (pg/ml)versus serum MIF (ng/ml) five hours following LPS/Galactosaminestimulation of female Balb/c mice.

FIGS. 11A–11B are bar graphs illustrating ELISA detection of IL-1β andTNF-α four hours following LPS (5 mg/kg) stimulation and the presence orabsence of 20 mg/kg body weight of compound 7e (i.p.).

FIGS. 12A–12C depict cumulative survival versus survival time (hours)(Kaplan-Meier Assessment of Survival) for Balb/c mice following i.p.dosing with 20 mg/kg of compound 7e or control vehicle at the time ofLPS (2 mg/kg (12A), 5 mg/kg (FIG. 12B), or 10 mg/kg (FIG. 12C)) andD-galactosamine (50 mg/kg) treatment. Each experiment included thirtymice with fifteen receiving the control vehicle and fifteen receivingthe compound of interest.

FIG. 13 is a graph illustrating survival time of 25% of mice versus LPSconcentration (mg; Sigma 055:B5) and D-galactosamine (50 mg/kg) in thepresence or absence of compound 7e (20 mg/kg body weight,). The datarepresent the averaging of six experiments using thirty mice each.

FIG. 14 represents the experimental protocol for testing MIF inhibitorsfor inhibiting arthritis in a collagen-induced arthritis mice model.Compound 7e was given two times a day for four days.

FIG. 15 is a bar graph illustrating caliper measurements of pawthickness as representative of paw edema on day 74. Values are expressedas the Mean+/−SEM for ten animals per group.

FIGS. 16A–16B are bar graphs depicting MIF (FIG. 16A) and TNF (FIG. 16B)levels in mouse sera of collagen induced-arthritic mice as measured byELISA. Values are expressed as the Mean+/−SEM of seven animals. Controlsare mice not treated with collagen or compound, CIA represents collageninduced arthritic mice, and compound 7e represents treated CIA mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

As an aid to understanding the preferred embodiments, certaindefinitions are provided herein.

The term “MIF activity,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to anactivity or effect mediated at least in part by macrophage migrationinhibitory factor. Accordingly, MIF activity includes, but is notlimited to, inhibition of macrophage migration, tautomerase activity(e.g., using phenylpyruvate or dopachrome), endotoxin induced shock,inflammation, glucocorticoid counter regulation, induction of thymidineincorporation into 3T3 fibroblasts, induction of erk phosphorylation andMAP kinase activity.

The term “export,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to ametabolically active process, which may or may not be energy-dependent,of transporting a translated cellular product to the cell membrane orthe extracellular space by a mechanism other than standard leadersequence directed secretion via a canonical leader sequence. Further,“export,” unlike secretion that is leader sequence-dependent, isresistant to brefeldin A (i.e., the exported protein is not transportedvia the ER/Golgi; brefeldin A is expected to have no direct effect ontrafficking of an exported protein) and other similar compounds. As usedherein, “export” may also be referred to as “non-classical secretion.”

The term “leaderless protein,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer to aprotein or polypeptide that lacks a canonical leader sequence, and isexported from inside a cell to the extracellular environment. Leaderlessproteins in the extracellular environment refer to proteins located inthe extracellular space, or associated with the outer surface of thecell membrane. Within the context of preferred embodiments, leaderlessproteins include naturally occurring proteins, such as macrophagemigration inhibitory factor and fragments thereof as well as proteinsthat are engineered to lack a leader sequence and are exported, orproteins that are engineered to include a fusion of a leaderlessprotein, or fraction thereof, with another protein.

The term “inhibitor,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a molecule(e.g., natural or synthetic compound) that can alter the conformation ofMIF and/or compete with a monoclonal antibody to MIF and decrease atleast one activity of MIF or its export from a cell as compared toactivity or export in the absence of the inhibitor. In other words, an“inhibitor” alters conformation and/or activity and/or export if thereis a statistically significant change in the amount of MIF measured, MIFactivity or in MIF protein detected extracellularly and/orintracellularly in an assay performed with an inhibitor, compared to theassay performed without the inhibitor.

The term “binding agent,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to anymolecule that binds MIF, including inhibitors.

In general, MIF inhibitors inhibit the physiological function of MIF,and thus are useful in the treatment of diseases where MIF may bepathogenic.

In certain of the preferred embodiments, inhibitors of MIF are providedthat have the following structures (Ia) and (Ib):

including stereoisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein: X is oxygen or sulfur; Y is —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, or —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1 or 2, with the proviso thatwhen n is 0, Z is —C(═O)—; R₁ is hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, or aminoalkyl R′R″N(CH₂)_(x)— wherein R′ andR″ are independently hydrogen, alkyl, substituted alkyl, aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, or dialkyl, and whereinx is 2 to 4; R₂ and R₃ are the same or different and are independently,halogen, —R₅, —OR₅, —SR₅ or —NR₅R₆; R₄ is —CH₂R₇, —C(═O)NR₅R₆,—C(═O)OR₇, —C(═O)R₇, or R₈; R₅ and R₆ are the same or different and areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl or substituted heterocyclealkyl; or R₅ andR₆ taken together with the nitrogen atom to which they are attached forma heterocycle or substituted heterocycle; R₇ is alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle,

heterocyclealkyl or substituted heterocyclealkyl; and R₈ is hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl orsubstituted heterocyclealkyl; with the provisos that: R₄ is not hydrogenor methyl when R₁ is phenyl, R₂ and R₃ are both hydrogen, X is oxygenand Y is —C(═O)OCH₂CH₃; R₄ is not methyl when R₁ is methyl, R₂ and R₃are both hydrogen, X is oxygen and Y is —NO₂; R₄ is not —CH₂CH₂OH whenR₁ is hydrogen or methyl, R₂ is 7-chloro, R₃ is hydrogen, X is oxygenand Y is —C(═O)OCH₂CH₃; and R₄ is not methyl when R₁ is methyl, R₂ ishydrogen or 7-chloro, R₃ is hydrogen, X is oxygen and Y is—C(═O)OCH₂CH₃. In certain embodiments, one or more of the provisos maynot apply.

In a preferred embodiment, methods are provided for reducing MIFactivity in a patient in need thereof by administering to the patient aneffective amount of a compound having the following structure (Ia)and/or (Ib):

including stereoisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein: X is oxygen or sulfur; Y is —NO, —NO₂, —C(═O)R₅,—C(═O)OR₅, —C(═O)NR₅R₆, —NR₅C(═O)R₅, —NR₅SO₂R₅, or —S(O)_(m)R₅; Z is—CH₂— or —C(═O)—; m is 0, 1, or 2; n is 0, 1 or 2, with the proviso thatwhen n is 0, Z is —C(═O)—; R₁ is hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl, dialkyl, or aminoalkyl R′R″N(CH₂)_(x)— wherein R′ andR″ are independently hydrogen, alkyl, substituted alkyl, aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, or dialkyl, and whereinx is 2 to 4; R₂ and R₃ are the same or different and are independently,halogen, —R₅, —OR₅, —SR₅ or —NR₅R₆; R₄ is —CH₂R₇, —C(═O)NR₅R₆,—C(═O)OR₇, —C(═O)R₇ or R₈; R₅ and R₆ are the same or different and areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl or substituted heterocyclealkyl; or R₅ andR₆ taken together with the nitrogen atom to which they are attached forma heterocycle or substituted heterocycle; R₇ is alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl; and R₈ is hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl; with the provisos that: R₄ is not hydrogen or methylwhen R₁ is phenyl, R₂ and R₃ are both hydrogen, X is oxygen and Y is—C(═O)OCH₂CH₃; R₄ is not methyl when R₁ is methyl, R₂ and R₃ are bothhydrogen, X is oxygen and Y is —NO₂; R₄ is not —CH₂CH₂OH when R₁ ishydrogen or methyl, R₂ is 7-chloro, R₃ is hydrogen, X is oxygen and Y is—C(═O)OCH₂CH₃; and R₄ is not methyl when R₁ is methyl, R₂ is hydrogen or7-chloro, R₃ is hydrogen, X is oxygen and Y is —C(═O)OCH₂CH₃. In certainembodiments, one or more of the provisos may not apply.

As used herein, the above terms have the following meanings. The term“alkyl,” as used herein is a broad term and is used in its ordinarysense, including, without limitation, to refer to a straight chain orbranched, noncyclic or cyclic, unsaturated or saturated aliphatichydrocarbon containing from 1 to 10 carbon atoms, while the term “loweralkyl” has the same meaning as alkyl but contains from 1 to 6 carbonatoms. Representative saturated straight chain alkyls include methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; whilesaturated branched alkyls include isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Representative saturated cyclicalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—CH₂cyclopropyl, —CH₂cyclobutyl, —CH₂cyclopentyl, —CH₂cyclohexyl, andthe like; while unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Cyclic alkyls, also referred to as“homocyclic rings,” and include di- and poly-homocyclic rings such asdecalin and adamantane. Unsaturated alkyls contain at least one doubleor triple bond between adjacent carbon atoms (referred to as an“alkenyl” or “alkynyl,” respectively). Representative straight chain andbranched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

The term “aryl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an aromaticcarbocyclic moiety such as phenyl or naphthyl.

The term “arylalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylhaving at least one alkyl hydrogen atoms replaced with an aryl moiety,such as benzyl, —CH₂(1 or 2-naphthyl), —(CH₂)₂phenyl, —(CH₂)₃phenyl,—CH(phenyl)₂, and the like.

The term “heteroalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to anaromatic heterocycle ring of 5- to 10 members and having at least oneheteroatom selected from nitrogen, oxygen and sulfur, and containing atleast 1 carbon atom, including both mono- and bicyclic ring systems.Representative heteroaryls include (but are not limited to) furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.

The term “heteroarylalkyl,” as used herein is a broad term and is usedin its ordinary sense, including, without limitation, to refer to analkyl having at least one alkyl hydrogen atom replaced with a heteroarylmoiety, such as —CH₂pyridinyl, —CH₂pyrimidinyl, and the like.

The terms “heterocycle” and “heterocycle ring,” as used herein, arebroad terms and are used in their ordinary sense, including, withoutlimitation, to refer to a 5- to 7-membered monocyclic, or 7- to14-membered polycyclic, heterocycle ring which is either saturated,unsaturated or aromatic, and which contains from 1 to 4 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzene ringas well as tricyclic (and higher) heterocyclic rings. The heterocyclemay be attached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined above. Thus, in addition to the aromaticheteroaryls listed above, heterocycles also include (but are not limitedto) morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heterocyclealkyl,” as used herein is a broad term and is usedin its ordinary sense, including, without limitation, to refer to analkyl having at least one alkyl hydrogen atom replaced with aheterocycle, such as —CH₂morpholinyl, and the like.

The term “substituted,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to any ofthe above groups (e.g., alkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, heterocycle or heterocyclealkyl) wherein at least onehydrogen atom is replaced with a substituent. In the case of a ketosubstituent (“—C(═O)—”) two hydrogen atoms are replaced. Whensubstituted, “substituents,” within the context of preferred embodiment,include halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino,alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,—NR_(a)R_(b), —NR_(a)C(═O)R_(b), —NR_(a)C(═O)NR_(a)R_(b),—NR_(a)C(═O)OR_(b) —NR_(a)SO₂R_(b), —OR_(a), —C(═O)R_(a) —C(═O)OR,—C(═O)N_(a)R_(b), —OC(═O)NR_(a)R_(b), —SH, —SR_(a), —SOR_(a),—S(═O)₂R_(a), —OS(═O)₂R_(a), —S(═O)₂OR_(a), wherein R_(a) and R_(b) arethe same or different and independently hydrogen, alkyl, haloalkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl or substituted heterocyclealkyl.

The term “halogen,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to fluoro,chloro, bromo and iodo.

The term “haloalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylhaving at least one hydrogen atom replaced with halogen, such astrifluoromethyl and the like.

The term “alkoxy,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylmoiety attached through an oxygen bridge (i.e., alkyl) such as methoxy,ethoxy, and the like.

The term “thioalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylmoiety attached through a sulfur bridge (i.e., —S-alkyl) such asmethylthio, ethylthio, and the like.

The term “alkylsulfonyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylmoiety attached through a sulfonyl bridge (i.e., —SO₂-alkyl) such asmethylsulfonyl, ethylsulfonyl, and the like.

The terms “alkylamino” and “dialkyl amino” as used herein, are broadterms and are used in their ordinary sense, including, withoutlimitation, to refer to one alkyl moiety or two alkyl moieties,respectively, attached through a nitrogen bridge (i.e., —N-alkyl) suchas methylamino, ethylamino, dimethylamino, diethylamino, and the like.

The term “hydroxyalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with at least one hydroxyl group.

The term “mono- or di(cycloalkyl)methyl,” as used herein is a broad termand is used in its ordinary sense, including, without limitation, torefer to a methyl group substituted with one or two cycloalkyl groups,such as cyclopropylmethyl, dicyclopropylmethyl, and the like.

The term “alkylcarbonylalkyl,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer toan alkyl substituted with a —C(═O)alkyl group.

The term “alkylcarbonyloxyalkyl,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer toan alkyl substituted with a —C(═O)Oalkyl group or a —OC(═O)alkyl group.

The term “alkyloxyalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with an —O-alkyl group.

The term “alkylthioalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with a —S-alkyl group.

The term “mono- or di(alkyl)amino,” as used herein is a broad term andis used in its ordinary sense, including, without limitation, to referto an amino substituted with one alkyl or with two alkyls, respectively.

The term “mono- or di(alkyl)aminoalkyl,” as used herein is a broad termand is used in its ordinary sense, including, without limitation, torefer to an alkyl substituted with a mono- or di(alkyl)amino.

The following numbering schemes are used in the context of preferredembodiments:

Depending upon the Z moiety, representative compounds of preferredembodiments include the following structure (II) when Z is methylene(—CH₂—) and structure (III) when Z is carbonyl (—C(═O)—):

In further embodiments, n is 0, 1, or 2 as represented by structures(IV), (V) and (VI), respectively:

In still further embodiments, compounds of preferred embodiments havethe following structure (VII) when X is oxygen and structure (VIII) whenX is sulfur:

Depending upon the Y group, compounds of preferred embodiments have thefollowing structures (IX) through (XIII):

The compounds of preferred embodiments may generally be employed as thefree acid or free base. Alternatively, the compounds of preferredembodiments may preferably be in the form of acid or base additionsalts. Acid addition salts of the free base amino compounds of preferredembodiments may be prepared by methods well known in the art, and may beformed from organic and inorganic acids. Suitable organic acids includemaleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic,mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids. Suitable inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric, and nitric acids. Base addition saltsof the free acid may similarly be prepared by methods well known in theart, and may be formed from suitable bases, such as cations chosen fromthe alkali and alkaline earth metals (e.g., lithium, sodium, potassium,magnesium, barium or calcium) as well as the ammonium cation. The term“pharmaceutically acceptable salt” of structure (Ia) or (Ib) is intendedto encompass any and all acceptable salt forms.

The compounds of structure (Ia) and (Ib) may be made according to theorganic synthesis techniques known to those skilled in this field, aswell as by the representative methods set forth in Example 1. Ingeneral, compounds of structure (Ia) may be made according to thefollowing Reaction Schemes.

In general, chloro intermediates of structure iii may be prepared fromthe corresponding alcohol ii by known techniques. The alcoholintermediate may, in turn, be prepared from starting material i byreaction with appropriate agents. Representative reactants andconditions are set forth in Example 1.

N-substituted piperazines of structure vi may be prepared bydeprotection of protected intermediate v (in this case, protected withN-tert-butyloxycarbonyl or “Boc” for purpose of illustration). Theprotected intermediate may be made from the N-protected piperazine iv byaddition of the desired R₄ group.

Intermediate iii from Reaction Scheme 1 may be reacted with intermediatevi from Reaction Scheme 3 to give compounds of preferred embodimentshaving structure (Ia).

Alternatively, intermediate iii of Reaction Scheme 1 may be reacted withprotected intermediate iva, to yield the protected reaction product vii.This protected reaction product may then be deprotected to yieldintermediate viii, followed by addition of the desired R₄ group to givecompounds of preferred embodiments having structure (Ia).

MIF as a Drug Target

Macrophage migration inhibitory factor (MIF) may be well suited foranalysis as a drug target as its activity has been implicated in avariety of pathophysiological conditions. For instance, MIF has beenshown to be a significant mediator in both inflammatory responses andcellular proliferation. In this regard, MIF has been shown to play rolesas a cytokine, a pituitary hormone, as glucocorticoid-inducedimmunomodulator, and just recently as a neuroimmunomodulator and inneuronal function. Takahashi et al, Mol. Med. 4:707–714, 1998; Bucala,Ann. N.Y. Acad. Sci. 840:74–82, 1998; Bacher et al., Mol. Med.4(4):217–230, 1998. Further, it has been recently demonstrated thatanti-MIF antibodies have a variety of uses, notably decreased tumorgrowth, along with an observed reduction in angiogenesis. Ogawa et al.,Cytokine 12(4):309–314, 2000; Metz and Bucala (supra). Accordingly,small molecules that can inhibit MIF have significant value in thetreatment of inflammatory responses, reduction of angiogenesis, viralinfection, bacterial infection, treatment of cancer (specificallytumorigenesis and apoptosis), treatment of graft versus host disease andassociated tissue rejection. A MIF inhibitor may be particularly usefulin a variety of immune related responses, tumor growth,glomerulonephritis, inflammation, malarial anemia, septic shock, tumorassociated angiogenesis, vitreoretinopathy, psoriasis, graft versus hostdisease (tissue rejection), atopic dermatitis, rheumatoid arthritis,inflammatory bowel disease, inflammatory lung disorders, otitis media,Crohn's disease, acute respiratory distress syndrome, delayed-typehypersensitivity. A MIF inhibitor may also be useful in the treatment ofstress and glucocorticoid function disorders, e.g., counter regulationof glucocorticoid action; or overriding of glucocorticoid mediatedsuppression of arachidonate release (Cys-60 based catalytic MIFoxidoreductase activity or JABI/CSNS-MIF-interaction based mechanism).

One example of the utility of a MIF inhibitor may be evidenced by thefact that following endotoxin exposure detectable serum concentrationsof MIF gradually increase during the acute phase (1–8 hours), peak at 8hours and persist during the post-acute phase (>8 hours) for up to 20hours. While not limited to any theory of operation, MIF may likely beproduced by activated T-cells and macrophages during the proinflammatorystage of endotoxin-induced shock, e.g., as part of the localizedresponse to infection. Once released by a pro-inflammatory stimulus,e.g., low concentrations of LPS, or by TNF-α and IFN-γ,macrophage-derived MIF may be the probable source of MIF produced duringthe acute phase of endotoxic shock. Both the pituitary, which releasesMIF in response to LPS, and macrophages are the probable source of MIFin the post-acute phase of endotoxic shock, when the infection is nolonger confined to a localized site. See, e.g., U.S. Pat. No. 6,080,407,incorporated herein by reference in its entirety and describing theseresults with anti-MIF antibodies.

As demonstrated herein, inhibitors of preferred embodiments inhibitlethality in mice following LPS challenge and likely attenuate IL-1β andTNF-α levels. Accordingly, a variety of inflammatory conditions may beamenable to treatment with a MIF inhibitor. In this regard, among otheradvantages, the inhibition of MIF activity and/or release may beemployed to treat inflammatory response and shock. Beneficial effectsmay be achieved by intervention at both early and late stages of theshock response. In this respect, while not limited to any theory ormechanism responsible for the protective effect of MIF inhibition,anti-MIF studies have demonstrated that introduction of anti-MIFantibodies is associated with an appreciable (up to 35–40%) reduction incirculating serum TNF-α levels. This reduction is consistent with theTNF-α-inducing activity of MIF on macrophages in vitro, and suggeststhat MIF may be responsible, in part, for the extremely high peak inserum TNF-α level that occurs 1–2 hours after endotoxin administrationdespite the fact that MIF cannot be detected in the circulation at thistime. Thus, MIF inhibition therapy may be beneficial at the early stagesof the inflammatory response.

MIF also plays a role during the post-acute stage of the shock response,and therefore, offers an opportunity to intervene at late stages whereother treatments, such as anti-TNF-α therapy, are ineffective.Inhibition of MIF can protect against lethal shock in animals challengedwith high concentrations of endotoxin (i.e., concentrations that inducerelease of pituitary MIF into the circulation), and in animalschallenged with TNF-α. Accordingly, the ability to inhibit MIF andprotect animals challenged with TNF indicates that neutralization of MIFduring the later, post-acute phase of septic shock may be efficacious.

As evidenced herein, TNF-α and IL-1β levels are correlated at least insome instances to MIF levels. Accordingly, an anti-MIF small moleculemay be useful in a variety of TNF-α and/or IL-1β associated diseasestates including transplant rejection, immune-mediated and inflammatoryelements of CNS disease (e.g., Alzheimer's, Parkinson's, multiplesclerosis, etc.), muscular dystrophy, diseases of hemostasis (e.g.,coagulopathy, veno occlusive diseases, etc.), allergic neuritis,granuloma, diabetes, graft versus host disease, chronic renal damage,alopecia (hair loss), acute pancreatitis, joint disease, congestiveheart failure, cardiovascular disease (restenosis, atherosclerosis),joint disease, and osteoarthritis.

Further, additional evidence in the art has indicated that steroidswhile potent inhibitors of cytokine production actually increase MIFexpression. Yang et al., Mol. Med. 4(6):413–424, 1998; Mitchell et al.,J. Biol. Chem. 274(25):18100–18106, 1999; Calandra and Bucala, Crit.Rev. Immunol. 17(1):77–88, 1997; Bucala, FASEB J. 10(14):1607–1613,1996. Accordingly, it may be of particular utility to utilize MIFinhibitors in combination with steroidal therapy for the treatment ofcytokine mediated pathophysiological conditions, such as inflammation,shock, and other cytokine-mediated pathological states, particularly inchronic inflammatory states such as rheumatoid arthritis. Suchcombination therapy may be beneficial even subsequent to the onset ofpathogenic or other inflammatory responses. For example, in the clinicalsetting, the administration of steroids subsequent to the onset ofseptic shock symptoms has proven of little benefit. See Bone et al., N.Engl. J. Med. 317: 653–658, 1987; Spring et al., N. Engl. J. Med. 311:1137–1141, 1984. Combination steroids/MIF inhibition therapy may beemployed to overcome this obstacle. Further, one of skill in the art mayunderstand that such therapies may be tailored to inhibit MIF releaseand/or activity locally and/or systemically.

Assays

The effectiveness of a compound as an inhibitor of MIF may be determinedby various assay methods. Suitable inhibitors of preferred embodimentsare capable of decreasing one or more activities associated with MIFand/or MIF export. A compound of structure (Ia) or (Ib) or any otherstructure may be assessed for activity as an inhibitor of MIF by one ormore generally accepted assays for this purpose, including (but notlimited to) the assays described below.

The assays may generally be divided into three categories, those being,assays which monitor export; those which monitor effector or smallmolecule binding, and those that monitor MIF activity. However, itshould be noted that combinations of these assays are within the scopeof the present application. Surprisingly, it appears that epitopemapping of MIF acts as surrogate for biological activity. For example,in one assay, the presence of a candidate inhibitor blocks the detectionof export of MIF from cells (e.g., THP-1 cells) measured using amonoclonal antibody such as that commercially available from R&D systems(Minneapolis, Minn.) whereas a polyclonal antibody demonstrates that MIFis present. Further, cellular based or in vitro assays may be employedto demonstrate that these potential inhibitors inhibit MIF activity. Inan alternative, these two assays (i.e., binding and activity assays) maybe combined into a singular assay which detects binding of a testcompound (e.g., the ability to displace monoclonal antibodies or inhibittheir binding) while also affecting MIF activity. Such assays includecombining an ELISA type assay (or similar binding assay) with a MIFtautomerism assay or similar functional assay. As one of ordinary skillin the art may readily recognize, the exact assay employed isirrelevant, provided it is able to detect the ability of the compound ofinterest to bind MIF. In addition, the assay preferably detects theability of the compound to inhibit a MIF activity because it selects forcompounds that interact with biologically active MIF and not inactiveMIF.

It should also be understood that compounds demonstrating the ability toinhibit monoclonal antibody binding to biologically active and notinactive MIF (e.g., small molecule inhibited), necessarily indicate thepresence of a compound (e.g., a small molecule) that is interacting withMIF either in a fashion which changes the conformation of MIF or blocksan epitope necessary for antibody binding. In other embodiments, MIFinhibitory activity may also be recognized as a consequence ofinterfering with the formation of a polypeptide complex that includesMIF; disturbing such a complex may result in a conformational changeinhibiting detection. Accordingly, the use of assays that monitorconformational changes in MIF, are advantageous when employed either inaddition to assays measuring competition between compounds, such assmall molecules with mAb or as a replacement of such an assay. A varietyof such assays are known in the art and include, calorimetry,circular-dichroism, fluorescence energy transfer, light-scattering,nuclear magnetic resonance (NMR), surface plasmon resonance,scintillation proximity assays (see U.S. Pat. No. 5,246,869), and thelike. See also WO02/07720-A1 and WO97/29635-A1. Accordingly, one ofskill in the art may recognize that any assay that indicates binding andpreferably conformational change or placement near the active site ofMIF may be utilized. Descriptions of several of the more complicatedproximity assays and conformational assays are set forth below, thisdiscussion is merely exemplary and in no way should be construed aslimiting to the type of techniques that may be utilized in preferredembodiments.

In one example, circular dichroism may be utilized to determinecandidate inhibitor binding. Circular dichroism (CD) is based in part onthe fact that most biological protein macromolecules are made up ofasymmetric monomer units, L-amino acids, so that they all possess theattribute of optical activity. Additionally, rigid structures like DNAor an alpha helical polypeptide have optical properties that can bemeasured using the appropriate spectroscopic system. In fact, largechanges in an easily measured spectroscopic parameter can provideselective means to identify conformational states and changes inconformational states under various circumstances, and sometimes toobserve the perturbation of single groups in or attached to themacromolecule. Further, CD analysis has been frequently employed toprobe the interactions of various macromolecules with small moleculesand ligands. See Durand et al., Eur. Biophys. J. 27(2):147–151, 1998;Kleifeld et al., Biochem 39(26):7702–7711, 2000; Bianchi et al., Biochem38(42):13844–13852, 1999; Sarver et al., Biochem Biophys Acta1434(2):304–316, 1999.

The Pasteur principle states that an optically active molecule must beasymmetric; that is, the molecule and its mirror image cannot besuperimposed. Plane polarized light is a combination of left circularlypolarized light and right circularly polarized light traveling in phase.The interaction of this light with an asymmetric molecule results in apreferential interaction of one circularly polarized component which, inan absorption region, is seen as a differential absorption (i.e., adichroism). See Urry, D. W., Spectroscopic Approaches to BiomolecularConformation, American Medical Association Press, Chicago, Ill., pp.33–120 (1969); Berova and Woody, Circular Dichroism: Principles andApplications, John Wiley & Sons, N.Y., (2000).

Circular dichroism, then, is an absorptive phenomenon that results whena chromophore interacts with plane polarized light at a specificwavelength. The absorption band can be either negative or positivedepending on the differential absorption of the right and leftcircularly polarized components for that chromophore. Unlike opticalrotatory dispersion (ORD) that measures the contributions of backgroundand the chromophore of interest many millimicrons from the region ofactual light interaction, CD offers the advantage of measuring opticalevents at the wavelength at which the event takes place. Circulardichroism, then, is specific to the electronic transition of thechromophore. See Berova and Woody, Circular Dichroism: Principles andApplications, John Wiley & Sons, N.Y., (2000).

Application of circular dichroism to solutions of macromolecules hasresulted in the ability to identify conformation states (Berova andWoody, Circular Dichroism: Principles and Applications, John Wiley &Sons, N.Y., (2000)). The technique can distinguish random coil, alphahelix, and beta chain conformation states of macromolecules. Inproteins, alpha helical fibrous proteins show absorption curves closelyresembling those of alpha helical polypeptides, but in globular proteinsof known structure, like lysozyme and ribonuclease, the helicalstructures are in rather poor agreement with X-ray crystallography work.A further source of difficulty in globular proteins is the prevalence ofaromatic chromophores on the molecules around 280 nm. An interestingexample of helical changes has been demonstrated using myoglobin andapomyoglobin. After removing the prosthetic group heme, the apoproteinremaining has a residual circular dichroic ellipticity reduced by 25%.This loss of helix is attributable to an uncoiling of 10–15 residues inthe molecule. Other non-peptide, optically active chromophores includetyrosine, tryptophan, phenylalanine, and cysteine when located in theprimary amino acid sequence of a macromolecule. Examples of non-peptideellipticities include the disulfide transition in ribonuclease and thecysteine transitions of insulin.

Accordingly, circular dichroism may be employed to screen candidateinhibitors for the ability to affect the conformation of MIF.

In certain embodiments provided herein, MIF-binding agent or inhibitorcomplex formation may be determined by detecting the presence of acomplex including MIF and a detectably labeled binding agent. Asdescribed in greater detail below, fluorescence energy signal detection,for example by fluorescence polarization, provides determination ofsignal levels that represent formation of a MIF-binding agent molecularcomplex. Accordingly, and as provided herein, fluorescence energysignal-based comparison of MIF-binding agent complex formation in theabsence and in the presence of a candidate inhibitor provides a methodfor identifying whether the agent alters the interaction between MIF andthe binding agent. For example, the binding agent may be a MIFsubstrate, an anti-MIF antibody, or a known inhibitor, while thecandidate inhibitor may be the compound to be tested or vice versa.

As noted above, certain preferred embodiments also pertain in part tofluorescence energy signal-based determination of MIF-binding agentcomplex formation. Fluorescence energy signal detection may be, forexample, by fluorescence polarization or by fluorescence resonanceenergy transfer, or by other fluorescence methods known in the art. Asan example of certain other embodiments, the MIF polypeptide may belabeled as well as the candidate inhibitor and may comprise an energytransfer molecule donor-acceptor pair, and the level of fluorescenceresonance energy transfer from energy donor to energy acceptor isdetermined.

In certain embodiments the candidate inhibitor and/or binding agent isdetectably labeled, and in particularly preferred embodiments thecandidate inhibitor and/or binding agent is capable of generating afluorescence energy signal. The candidate inhibitor and/or binding agentcan be detectably labeled by covalently or non-covalently attaching asuitable reporter molecule or moiety, for example any of variousfluorescent materials (e.g., a fluorophore) selected according to theparticular fluorescence energy technique to be employed, as known in theart and based upon the methods described herein. Fluorescent reportermoieties and methods for as provided herein can be found, for example inHaugland (1996 Handbook of Fluorescent Probes and ResearchChemicals—Sixth Ed., Molecular Probes, Eugene, Oreg.; 1999 Handbook ofFluorescent Probes and Research Chemicals—Seventh Ed., Molecular Probes,Eugene, Oreg., http://www.probes.com/lit/) and in references citedtherein. Particularly preferred for use as such a fluorophore inpreferred embodiments are fluorescein, rhodamine, Texas Red,AlexaFluor-594, AlexaFluor-488, Oregon Green, BODIPY-FL, and Cy-5.However, any suitable fluorophore may be employed, and in certainembodiments fluorophores other than those listed may be preferred.

As provided herein, a fluorescence energy signal includes anyfluorescence emission, excitation, energy transfer, quenching, ordequenching event or the like. Typically a fluorescence energy signalmay be mediated by a fluorescent detectably labeled candidate inhibitorand/or binding agent in response to light of an appropriate wavelength.Briefly, and without wishing to be bound by theory, generation of afluorescence energy signal generally involves excitation of afluorophore by an appropriate energy source (e.g., light of a suitablewavelength for the selected fluorescent reporter moiety, or fluorophore)that transiently raises the energy state of the fluorophore from aground state to an excited state. The excited fluorophore in turn emitsenergy in the form of detectable light typically having a different(e.g., usually longer) wavelength from that preferred for excitation,and in so doing returns to its energetic ground state. The methods ofpreferred embodiments contemplate the use of any fluorescence energysignal, depending on the particular fluorophore, substrate labelingmethod and detection instrumentation, which may be selected readily andwithout undue experimentation according to criteria with which thosehaving ordinary skill in the art are familiar.

In certain embodiments, the fluorescence energy signal is a fluorescencepolarization (FP) signal. In certain other embodiments, the fluorescenceenergy signal may be a fluorescence resonance energy transfer (FRET)signal. In certain other preferred embodiments the fluorescence energysignal can be a fluorescence quenching (FQ) signal or a fluorescenceresonance spectroscopy (FRS) signal. (For details regarding FP, FRET, FQand FRS, see, for example, WO97/39326; WO99/29894; Haugland, Handbook ofFluorescent Probes and Research Chemicals—6th Ed., 1996, MolecularProbes, Inc., Eugene, Oreg., p. 456; and references cited therein.)

FP, a measurement of the average angular displacement (due to molecularrotational diffusion) of a fluorophore that occurs between itsabsorption of a photon from an energy source and its subsequent emissionof a photon, depends on the extent and rate of rotational diffusionduring the excited state of the fluorophore, on molecular size andshape, on solution viscosity and on solution temperature (Perrin, 1926J. Phys. Rad. 1:390). When viscosity and temperature are held constant,FP is directly related to the apparent molecular volume or size of thefluorophore. The polarization value is a ratio of fluorescenceintensities measured in distinct planes (e.g., vertical and horizontal)and is therefore a dimensionless quantity that is unaffected by theintensity of the fluorophore. Low molecular weight fluorophores, such asthe detectably labeled candidate inhibitor and/or binding agent providedherein, are capable of rapid molecular rotation in solution (i.e., lowanisotropy) and thus give rise to low fluorescence polarizationreadings. When complexed to a higher molecular weight molecule such asMIF as provided herein, however, the fluorophore moiety of the substrateassociates with a complex that exhibits relatively slow molecularrotation in solution (i.e., high anisotropy), resulting in higherfluorescence polarization readings.

This difference in the polarization value of free detectably labeledcandidate inhibitor and/or binding agent compared to the polarizationvalue of MIF:candidate inhibitor and/or binding agent complex may beemployed to determine the ratio of complexed (e.g., bound) to free. Thisdifference may also be employed to detect the influence of a candidateagent (i.e., candidate inhibitor) on the formation of such complexesand/or on the stability of a pre-formed complex, for example bycomparing FP detected in the absence of an agent to FP detected in thepresence of the agent. FP measurements can be performed withoutseparation of reaction components.

As noted above, one aspect of a preferred embodiment utilizes thebinding or displacement of a monoclonal antibody, known inhibitor, orother binding agent and/or complex formation of the candidate inhibitorwith MIF to provide a method of identifying an inhibitor that alters theactivity of MIF. Surprisingly, the inhibitors of preferred embodimentswere identified in such a nonconventional manner. In this regard, aclass of compounds demonstrated the ability to inhibit/decreasemonoclonal antibody binding to a biologically active MIF that isnaturally produced from cells while not affecting the antibody's abilityto recognize inactive (recombinant) MIF (as is available from commercialsources) and also demonstrated pronounced modulation of MIF activity invivo. Accordingly, antibody binding may be preferred as a surrogate forenzyme activity, thus eliminating the need to run expensive and complexenzymatic assays on each candidate compound. As those of ordinary skillin the art readily appreciate, the ability to avoid enzymatic assaysleads to an assay that may be extremely well suited for high throughputuse.

Further, as those of ordinary skill in the art can readily appreciate,such an assay may be employed outside of the MIF context and whereverenzyme or biological activity can be replaced by a binding assay. Forexample, any enzyme or other polypeptide whose biologically active formis recognized by a monoclonal antibody that does not recognize theinactive form (e.g., small molecule inhibited form) may be preferred.Within the context of an enzyme, the monoclonal antibody may bind theactive site, but be displaced by a small molecule. Thus, any smallmolecule that displaces the antibody may be a strong lead as a potentialenzyme inhibitor. As those of skill in the art appreciate, the antibodymay recognize an epitope that changes conformation depending on theactive state of the enzyme, and that binding of a small molecule suchthat it precludes antibody binding to this epitope may also act as asurrogate for enzymatic activity even though the epitope may not be atthe active site. Accordingly, the type of assay utilized herein may beexpanded to be employed with essentially any polypeptide whereinantibody displacement is predictive of activity loss. Thus, in itssimplest form any polypeptide, e.g., enzyme and its associatedneutralizing antibody may be employed to screen for small molecules thatdisplace this antibody, thereby identifying likely inhibitors.

A MIF-binding agent/candidate inhibitor complex may be identified by anyof a variety of techniques known in the art for demonstrating anintermolecular interaction between MIF and another molecule as describedabove, for example, co-purification, co-precipitation,co-immunoprecipitation, radiometric or fluorimetric assays, westernimmunoblot analyses, affinity capture including affinity techniques suchas solid-phase ligand-counterligand sorbent techniques, affinitychromatography and surface affinity plasmon resonance, NMR, and the like(see, e.g., U.S. Pat. No. 5,352,660). Determination of the presence ofsuch a complex may employ antibodies, including monoclonal, polyclonal,chimeric and single-chain antibodies, and the like, that specificallybind to MIF or the binding agent.

Labeled MIF and/or labeled binding agents/candidate inhibitors can alsobe employed to detect the presence of a complex. The molecule ofinterest can be labeled by covalently or non-covalently attaching asuitable reporter molecule or moiety, for example any of variousenzymes, fluorescent materials, luminescent materials, and radioactivematerials. Examples of suitable enzymes include, but are not limited to,horseradish peroxidase, alkaline phosphatase, β-galactosidase, andacetylcholinesterase. Examples of suitable fluorescent materialsinclude, but are not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, phycoerythrin, Texas Red, AlexaFluor-594, AlexaFluor-488,Oregon Green, BODIPY-FL and Cy-5. Appropriate luminescent materialsinclude, but are not limited to, luminol and suitable radioactivematerials include radioactive phosphorus [³²P], iodine [¹²⁵I or ¹³¹I] ortritium [³H].

MIF and the binding agent and/or the candidate inhibitor are combinedunder conditions and for a time sufficient to permit formation of anintermolecular complex between the components. Suitable conditions forformation of such complexes are known in the art and can be readilydetermined based on teachings provided herein, including solutionconditions and methods for detecting the presence of a complex and/orfor detecting free substrate in solution.

Association of a detectably labeled binding agent(s) and/or candidateinhibitor(s) in a complex with MIF, and/or binding agent or candidateinhibitor that is not part of such a complex, may be identifiedaccording to a preferred embodiment by detection of a fluorescenceenergy signal generated by the substrate. Typically, an energy sourcefor detecting a fluorescence energy signal is selected according tocriteria with which those having ordinary skill in the art are familiar,depending on the fluorescent reporter moiety with which the substrate islabeled. The test solution, containing (a) MIF and (b) the detectablylabeled binding agent and/or candidate inhibitor, is exposed to theenergy source to generate a fluorescence energy signal, which isdetected by any of a variety of well known instruments and identifiedaccording to the particular fluorescence energy signal. In preferredembodiments, the fluorescence energy signal is a fluorescencepolarization signal that can be detected using a spectrofluorimeterequipped with polarizing filters. In particularly preferred embodimentsthe fluorescence polarization assay is performed simultaneously in eachof a plurality of reaction chambers that can be read using an LJLCRITERION™ Analyst (LJL Biosystems, Sunnyvale, Calif.) plate reader, forexample, to provide a high throughput screen (HTS) having variedreaction components or conditions among the various reaction chambers.Examples of other suitable instruments for obtaining fluorescencepolarization readings include the POLARSTAR™ (BMG Lab Technologies,Offenburg, Germany), BEACON™ (Panvera, Inc., Madison, Wis.) and thePOLARION™ (Tecan, Inc., Research Triangle Park, NC) devices.

Determination of the presence of a complex that has formed between MIFand a binding agent and/or a candidate inhibitor may be performed by avariety of methods, as noted above, including fluorescence energy signalmethodology as provided herein and as known in the art. Suchmethodologies may include, by way of illustration and not limitation FP,FRET, FQ, other fluorimetric assays, co-purification, co-precipitation,co-immunoprecipitation, radiometric, western immunoblot analyses,affinity capture including affinity techniques such as solid-phaseligand-counterligand sorbent techniques, affinity chromatography andsurface affinity plasmon resonance, circular dichroism, and the like.For these and other useful affinity techniques, see, for example,Scopes, R. K., Protein Purification: Principles and Practice, 1987,Springer-Verlag, NY; Weir, D. M., Handbook of Experimental Immunology,1986, Blackwell Scientific, Boston; and Hermanson, G. T. et al.,Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc.,California; which are hereby incorporated by reference in theirentireties, for details regarding techniques for isolating andcharacterizing complexes, including affinity techniques. In variousembodiments, MIF may interact with a binding agent and/or candidateinhibitor via specific binding if MIF binds the binding agent and/orcandidate inhibitor with a K_(a) of greater than or equal to about 10⁴M⁻¹, preferably of greater than or equal to about 10⁵ M⁻¹, morepreferably of greater than or equal to about 10⁶ M⁻¹ and still morepreferably of greater than or equal to about 10⁷ M⁻¹ to 10¹¹ M⁻¹.Affinities of binding partners can be readily calculated from datagenerated according to the fluorescence energy signal methodologiesdescribed above and using conventional data handling techniques, forexample, those described by Scatchard et al., Ann. N.Y. Acad. Sci 51:660(1949).

For example, in various embodiments where the fluorescence energy signalis a fluorescence polarization signal, fluorescence anisotropy (inpolarized light) of the free detectably labeled candidate inhibitorand/or binding agent can be determined in the absence of MIF, andfluorescence anisotropy (in polarized light) of the fully boundsubstrate can be determined in the presence of a titrated amount of MIF.Fluorescence anisotropy in polarized light varies as a function of theamount of rotational motion that the labeled candidate inhibitor and/orbinding agent undergoes during the lifetime of the excited state of thefluorophore, such that the anisotropies of free and fully boundcandidate inhibitor and/or binding agent can be usefully employed todetermine the fraction of candidate inhibitor and/or binding agent boundto MIF in a given set of experimental conditions, for instance, thosewherein a candidate agent is present (see, e.g., Lundblad et al., 1996Molec. Endocrinol. 10:607; Dandliker et al., 1971 Immunochem. 7:799;Collett, E., Polarized Light: Fundamentals and Applications, 1993 MarcelDekker, New York).

Certain of the preferred embodiments pertain in part to the use ofintermolecular energy transfer to monitor MIF-binding agent complexformation and stability and/or MIF-candidate inhibitor complexformation.

Energy transfer (ET) is generated from a resonance interaction betweentwo molecules: an energy-contributing “donor” molecule and anenergy-receiving “acceptor” molecule. Energy transfer can occur when (1)the emission spectrum of the donor overlaps the absorption spectrum ofthe acceptor and (2) the donor and the acceptor are within a certaindistance (for example, less than about 10 nm) of one another. Theefficiency of energy transfer is dictated largely by the proximity ofthe donor and acceptor, and decreases as a power of 6 with distance.Measurements of ET thus strongly reflect the proximity of the acceptorand donor compounds, and changes in ET sensitively reflect changes inthe proximity of the compounds such as, for example, association ordissociation of the donor and acceptor.

It is therefore an aspect of a preferred embodiment to provide a methodfor assaying a candidate MIF inhibitor, in pertinent part, by contactingMIF or an MIF-binding agent complex including one or more ET donor andan ET acceptor molecules, exciting the ET donor to produce an excited ETdonor molecule and detecting a signal generated by energy transfer fromthe ET donor to the ET acceptor. As also provided herein, the method canemploy any suitable ET donor molecule and ET acceptor molecule that canfunction as a donor-acceptor pair.

In certain preferred embodiments, a detectable signal that is generatedby energy transfer between ET donor and acceptor molecules results fromfluorescence resonance energy transfer (FRET), as discussed above. FREToccurs within a molecule, or between two different types of molecules,when energy from an excited donor fluorophore is transferred directly toan acceptor fluorophore (for a review, see Wu et al., AnalyticalBiochem. 218:1–13, 1994).

In other aspects of preferred embodiments, the ability of a candidateinhibitor to effect MIF export is tested.

The first step of such an assay is performed to detect MIFextracellularly. For this assay, test cells expressing MIF are employed(e.g., THP-1 cells). Either the test cells may naturally produce theprotein or produce it from a transfected expression vector. Test cellspreferably normally express the protein, such that transfection merelyincreases expressed levels. Thus, for expression of MIF and IL-1, THP1cells are preferred. When one is assaying virally-derived proteins, suchas HIV tat, if the test cells do not “naturally” produce the protein,they may readily be transfected using an appropriate vector, so that thetest cells express the desired protein, as those of skill in the artreadily appreciate.

Following expression, MIF is detected by any one of a variety ofwell-known methods and procedures. Such methods include staining withantibodies in conjunction with flow cytometry, confocal microscopy,image analysis, immunoprecipitation of cell cytosol or medium, Westernblot of cell medium, ELISA, 1- or 2-D gel analysis, HPLC, or bioassay. Aconvenient assay for initial screening is ELISA. MIF export may beconfirmed by one of the other assays, preferably by immunoprecipitationof cell medium following metabolic labeling. Briefly, cells expressingMIF protein are pulse labeled for 15 minutes with ³⁵S-methionine and/or³⁵S-cysteine in methionine and/or cysteine free medium and chased inmedium supplemented with excess methionine and/or cysteine. Mediafractions are collected and clarified by centrifugation, such as in amicrofuge. Lysis buffer containing 1% NP-40, 0.5% deoxycholate (DOC), 20mM Tris, pH 7.5, 5 mM EDTA, 2 mM EGTA, 10 nM PMSF, 10 ng/ml aprotinin,10 ng/ml leupeptin, and 10 ng/ml pepstatin is added to the clarifiedmedium. An antibody to MIF is added and following incubation in thecold, a precipitating second antibody or immunoglobulin binding protein,such as protein A-Sepharose® or GammaBind™-Sepharose®, is added forfurther incubation. In parallel, as a control, a cytosolic protein ismonitored and an antibody to the cytosolic protein is preferred inimmunoprecipitations. Immune complexes are pelleted and washed withice-cold lysis buffer. Complexes are further washed with ice-cold IPbuffer (0.15 M NaCl, 10 mM Na-phosphate, pH 7.2, 1% DOC, 1% NP-40, 0.1%SDS). Immune complexes are eluted directly into SDS-gel sample bufferand electrophoresed in SDS-PAGE. The gel is processed for fluorography,dried and exposed to X-ray film. Alternatively cells can be engineeredto produced a MIF that is tagged with a reporter so that the presence ofan active MIF can be through the surrogate activity of the reporter.

While not wishing to be bound to theory, it is believed that the presentinhibitors function by interacting directly with the naturally producedMIF complex in such a fashion as to alter the protein's conformationenough to block its biological activity. This interaction can be mappedby X-ray crystallography of MIF-compound co-crystals to determine theexact site of interaction. One site localizes to the pocket that isresponsible for the tautomerase activity of MIF.

Screening assays for inhibitors of MIF export varies according to thetype of inhibitor and the nature of the activity that is being affected.Assays may be performed in vitro or in vivo. In general, in vitro assaysare designed to evaluate MIF activity, or multimerization, and in vivoassays are designed to evaluate MIF activity, extracellular, andintracellular localization in a model cell or animal system. In any ofthe assays, a statistically significant increase or decrease compared toa proper control is indicative of enhancement or inhibition.

One in vitro assay can be performed by examining the effect of acandidate compound on the ability of MIF to inhibit macrophagemigration. Briefly, human peripheral blood monocytes are preferred asindicator cells in an agarose-droplet assay system essentially asdescribed by Weiser et al., Cell. Immunol. 90:167–178, 1985 andHarrington et al., J. Immunol. 110:752–759, 1973. Other assay systems ofanalyzing macrophage migration may also be employed. Such an assay isdescribed by Hermanowski-Vosatka et al., Biochem. 38:12841–12849, 1999.

An alternative in vitro assay is designed to measure the ability of MIFto catalyze tautomerization of the D-isomer of dopachrome (see Rosengrenet al., Mol. Med. 2:143–149, 1996; Winder et al., J. Cell Sci.106:153–166, 1993; Aroca et al., Biochem. J. 277:393–397). Briefly, inthis method, D-dopachrome is provided to MIF in the presence and absenceof a candidate inhibitor and the ability to catalyze the tautomerizationto 5,6-dihydroxyindole-2-carboxylic acid (DHICA) is monitored. However,use of methyl esters of D-dopachrome may be preferred in that a fasterreaction rate is observed. Detection of the tautomerization can beperformed by any one of a variety of standard methods.

In a similar assay, the ability of MIF to catalyze the tautomerizationof phenylpyruvate may be tested (see Johnson et al., Biochem.38(48):16024–16033, 1999). Briefly, in this method, typicallyketonization of phenylpyruvate or (p-hydroxyphenyl)pyruvate is followedby spectroscopy. Further, product formation may be verified by treatmentof these compounds with MIF and subsequent conversion to malate for ¹HNMR analysis.

In vivo assays can be performed in cells transfected either transientlyor stably with an expression vector containing a MIF nucleic acidmolecule, such as those described herein. These cells are preferred tomeasure MIF activity (e.g., modulation of apoptosis, proliferation,etc.) or extracellular and intracellular localization in the presence orabsence of a candidate compound. When assaying for apoptosis, a varietyof cell analyses may be employed including, for example, dye stainingand microscopy to examine nucleic acid fragmentation and porosity of thecells.

Other assays may be performed in model cell or animal systems, byproviding to the system a recombinant or naturally occurring form of MIFor inducing endogenous MIF expression in the presence or absence of testcompound, thereby determining a statistically significant increase ordecrease in the pathology of that system. For example, LPS can beemployed to induce a toxic shock response.

The assays briefly described herein may be employed to identify aninhibitor that is specific for MIF.

In any of the assays described herein, a test cell may express the MIFnaturally (e.g., THP-1 cells) or following introduction of a recombinantDNA molecule encoding the protein. Transfection and transformationprotocols are well known in the art and include Ca₂PO₄-mediatedtransfection, electroporation, infection with a viral vector,DEAE-dextran mediated transfection, and the like. As an alternative tothe proteins described above, chimeric MIF proteins (i.e., fusion of MIFprotein with another protein or protein fragment), or protein sequencesengineered to lack a leader sequence may be employed. In a similarfashion, a fusion may be constructed to direct secretion, export, orcytosolic retention. Any and all of these sequences may be employed in afusion construct with MIF to assist in assaying inhibitors. The hostcell can also express MIF as a result of being diseased, infected with avirus, and the like. Secreted proteins that are exported by virtue of aleader sequence are well known and include, human chorionic gonadatropin(hCGα), growth hormone, hepatocyte growth factor, transferrin, nervegrowth factor, vascular endothelial growth factor, ovalbumin, andinsulin-like growth factor. Similarly, cytosolic proteins are well knownand include, neomycin phosphotransferase, β-galactosidase, actin andother cytoskeletal proteins, and enzymes, such as protein kinase A or C.The most useful cytosolic or secreted proteins are those that arereadily measured in a convenient assay, such as ELISA. The threeproteins (leaderless, secreted, and cytosolic) may be co-expressednaturally, by co-transfection in the test cells, or transfectedseparately into separate host cells. Furthermore, for the assaysdescribed herein, cells may be stably transformed or express the proteintransiently.

Immunoprecipitation is one such assay that may be employed to determineinhibition. Briefly, cells expressing MIF naturally or from anintroduced vector construct are labeled with ³⁵S-methionine and/or³⁵S-cysteine for a brief period of time, typically 15 minutes or longer,in methionine- and/or cysteine-free cell culture medium. Following pulselabeling, cells are washed with medium supplemented with excessunlabeled methionine and cysteine plus heparin if the leaderless proteinis heparin binding. Cells are then cultured in the same chase medium forvarious periods of time. Candidate inhibitors or enhancers are added tocultures at various concentration. Culture supernatant is collected andclarified. Supernatants are incubated with anti-MIF immune serum or amonoclonal antibody, or with anti-tag antibody if a peptide tag ispresent, followed by a developing reagent such as Staphylococcus aureusCowan strain 1, protein A-Sepharose®, or Gamma-bind™ G-Sepharose®.Immune complexes are pelleted by centrifugation, washed in a buffercontaining 1% NP-40 and 0.5% deoxycholate, EGTA, PMSF, aprotinin,leupeptin, and pepstatin. Precipitates are then washed in a buffercontaining sodium phosphate pH 7.2, deoxycholate, NP-40, and SDS. Immunecomplexes are eluted into an SDS gel sample buffer and separated bySDS-PAGE. The gel is processed for fluorography, dried, and exposed tox-ray film.

Alternatively, ELISA may be preferred to detect and quantify the amountof MIF in cell supernatants. ELISA is preferred for detection in highthroughput screening. Briefly, 96-well plates are coated with ananti-MIF antibody or anti-tag antibody, washed, and blocked with 2% BSA.Cell supernatant is then added to the wells. Following incubation andwashing, a second antibody (e.g., to MIF) is added. The second antibodymay be coupled to a label or detecting reagent, such as an enzyme or tobiotin. Following further incubation, a developing reagent is added andthe amount of MIF determined using an ELISA plate reader. The developingreagent is a substrate for the enzyme coupled to the second antibody(typically an anti-isotype antibody) or for the enzyme coupled tostreptavidin. Suitable enzymes are well known in the art and includehorseradish peroxidase, which acts upon a substrate (e.g., ABTS)resulting in a colorimetric reaction. It is recognized that rather thanusing a second antibody coupled to an enzyme, the anti-MIF antibody maybe directly coupled to the horseradish peroxidase, or other equivalentdetection reagent. If desired, cell supernatants may be concentrated toraise the detection level. Further, detection methods, such as ELISA andthe like may be employed to monitor intracellular as well asextracellular levels of MIF. When intracellular levels are desired, acell lysate is preferred. When extracellular levels are desired, mediacan be screened.

ELISA may also be readily adapted for screening multiple candidateinhibitors or enhancers with high throughput. Briefly, such an assay isconveniently cell based and performed in 96-well plates. Test cells thatnaturally or stably express MIF are plated at a level sufficient forexpressed product detection, such as, about 20,000 cells/well. However,if the cells do not naturally express the protein, transient expressionis achieved, such as by electroporation or Ca₂PO₄-mediated transfection.For electroporation, 100 μl of a mixture of cells (e.g., 150,000cells/ml) and vector DNA (5 μg/ml) is dispensed into individual wells ofa 96-well plate. The cells are electroporated using an apparatus with a96-well electrode (e.g., ECM 600 Electroporation System, BTX,Genetronics, Inc.). Optimal conditions for electroporation areexperimentally determined for the particular host cell type. Voltage,resistance, and pulse length are the typical parameters varied.Guidelines for optimizing electroporation may be obtained frommanufacturers or found in protocol manuals, such as Current Protocols inMolecular Biology (Ausubel et al. (ed.), Wiley Interscience, 1987).Cells are diluted with an equal volume of medium and incubated for 48hours. Electroporation may be performed on various cell types, includingmammalian cells, yeast cells, bacteria, and the like. Followingincubation, medium with or without inhibitor is added and cells arefurther incubated for 1–2 days. At this time, culture medium isharvested and the protein is assayed by any of the assays herein.Preferably, ELISA is employed to detect the protein. An initialconcentration of 50 μM is tested. If this amount gives a statisticallysignificant reduction of export or reduction of monoclonal Ab detection,the candidate inhibitor is further tested in a dose response.

Alternatively, concentrated supernatant may be electrophoresed on anSDS-PAGE gel and transferred to a solid support, such as nylon ornitrocellulose. MIF is then detected by an immunoblot (see Harlow,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988),using an antibody to MIF containing an isotopic or non-isotopic reportergroup. These reporter groups include, but are not limited to enzymes,cofactors, dyes, radioisotopes, luminescent molecules, fluorescentmolecules, and biotin. Preferably, the reporter group is ¹²⁵I orhorseradish peroxidase, which may be detected by incubation with2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid. These detectionassays described above are readily adapted for use if MIF contains apeptide tag. In such case, the antibody binds to the peptide tag. Otherassays include size or charge-based chromatography, including HPLC, andaffinity chromatography.

Alternatively, a bioassay may be employed to quantify the amount ofactive MIF present in the cell medium. For example, the bioactivity ofthe MIF may be measured by a macrophage migration assay. Briefly, cellstransfected with an expression vector containing MIF are cultured forapproximately 30 hours, during which time a candidate inhibitor orenhancer is added. Following incubation, cells are transferred to a lowserum medium for a further 16 hours of incubation. The medium is removedand clarified by centrifugation. A lysis buffer containing proteaseinhibitors is added to the medium or, in the alternative, cells arelysed and internal levels are determined as follows. Bioactivity of MIFis then measured by macrophage migration assay, isomerase activity, or aproliferation assay. A proliferation assay is performed by addingvarious amounts of the eluate to cultured quiescent 3T3 cells. Tritiatedthymidine is added to the medium and TCA precipitable counts aremeasured approximately 24 hours later. Reduction of the vital dye MTT isan alternative way to measure proliferation. For a standard, purifiedrecombinant human FGF-2 may be employed. Other functions may be assayedin other appropriate bioassays available in the art, such as CPS inducedtoxic shock, TSST-1 induced toxic shock, collagen induced arthritis,etc.

Other in vitro angiogenic assays include bioassays that measureproliferation of endothelial cells within collagen gel (Goto et al., LabInvest. 69:508, 1993), co-culture of brain capillary endothelial cellson collagen gels separated by a chamber from cells exporting the MIFprotein (Okamure et al., B.B.R.C. 186:1471, 1992; Abe et al., J. Clin.Invest. 92:54, 1993), or a cell migration assay (see Warren et al., J.Clin. Invest. 95:1789, 1995).

Production of Antibodies

The term “antibody,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to polyclonal,monospecific, and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. With regard to an anti-MIF/target antibodyof preferred embodiments, the term “antigen” as used herein is a broadterm and is used in its ordinary sense, including, without limitation,to refer to a macrophage migration inhibitory factor polypeptide or atarget polypeptide, variant, or functional fragment thereof. Ananti-MIF/target antibody, or antigen binding fragment of such anantibody, may be characterized as having specific binding activity forthe target polypeptide or epitope thereof of at least about 1×10⁵ M⁻¹,generally at least about 1×10⁶ M⁻¹, and preferably at least about 1×10⁸M⁻¹. Fab, F(ab′)₂, Fd and Fv fragments of an anti-MIF/target antibody,which retain specific binding activity for a MIF/targetpolypeptide-specific epitope, are encompassed within preferredembodiments. Of particular interest are those antibodies that bindactive polypeptides and are displaced upon binding of an inhibitorysmall molecule. Those of skill in the art readily appreciate that suchdisplacement can be the result of a conformational change, thus changingthe nature of the epitope, competitive binding with the epitope, orsteric exclusion of the antibody from its epitope. In one example, theactive site of an enzyme may be the epitope for a particular antibodyand upon binding of a small molecule at or near the active site,immunoreactivity of the antibody is lost, thereby allowing the use ofloss of immunoreactivity with an antibody as a surrogate marker forenzyme activity.

In addition, the term “antibody” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer tonaturally occurring antibodies as well as non-naturally occurringantibodies, including, for example, single chain antibodies, chimeric,bifunctional and humanized antibodies, as well as antigen-bindingfragments thereof. Such non-naturally occurring antibodies may beconstructed using solid phase peptide synthesis, may be producedrecombinantly, or may be obtained, for example, by screeningcombinatorial libraries including variable heavy chains and variablelight chains (Huse et al., Science 246:1275–1281 (1989)). These andother methods of making, for example, chimeric, humanized, CDR-grafted,single chain, and bifunctional antibodies are well known in the art(Winter and Harris, Immunol. Today 14:243–246 (1993); Ward et al.,Nature 341:544–546 (1989); Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1992); Borrabeck,Antibody Engineering, 2d ed., Oxford Univ. Press (1995); Hilyard et al.,Protein Engineering: A practical approach, IRL Press (1992)).

In certain preferred embodiments, an anti-MIF/target antibody may beraised using as an immunogen such as, for example, an isolated peptideincluding the active site region of MIF or the target polypeptide, whichcan be prepared from natural sources or produced recombinantly, asdescribed above, or an immunogenic fragment of a MIF/target polypeptide(e.g., immunogenic sequences including 8–30 or more contiguous aminoacid sequences), including synthetic peptides, as described above. Anon-immunogenic peptide portion of a MIF/target polypeptide can be madeimmunogenic by coupling the hapten to a carrier molecule such as bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressingthe peptide portion as a fusion protein. Various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art (Harlow and Lane, supra, 1992).

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse, or other mammal, are well known in the art. In addition,monoclonal antibodies may be obtained using methods that are well knownand routine in the art (Harlow and Lane, supra, 1992). For example,spleen cells from a target polypeptide-immunized mammal can be fused toan appropriate myeloma cell line such as SP/02 myeloma cells to producehybridoma cells. Cloned hybridoma cell lines may be screened using alabeled target polypeptide or functional fragment thereof to identifyclones that secrete target polypeptide monoclonal antibodies having thedesired specificity. Hybridomas expressing target polypeptide monoclonalantibodies having a desirable specificity and affinity may be isolatedand utilized as a continuous source of the antibodies, which are useful,for example, for preparing standardized kits. Similarly, a recombinantphage that expresses, for example, a single chain anti-targetpolypeptide also provides a monoclonal antibody that may be employed forpreparing standardized kits.

Applications and Methods Utilizing Inhibitors of MIF

Inhibitors of MIF have a variety of applicable uses, as noted above.Candidate inhibitors of MIF may be isolated or procured from a varietyof sources, such as bacteria, fungi, plants, parasites, libraries ofchemicals (small molecules), peptides or peptide derivatives and thelike. Further, one of skill in the art recognize that inhibition hasoccurred when a statistically significant variation from control levelsis observed.

Given the various roles of MIF in pathology and homeostasis, inhibitionof MIF activity or MIF extracellular localization may have a therapeuticeffect. For example, recent studies have demonstrated that MIF is amediator of endotoxemia, where anti-MIF antibodies fully protected micefrom LPS-induced lethality. See Bernhagen et al., Nature 365:756–759,1993; Calandra et al., J. Exp. Med. 179:1895–1902, 1994; Bernhagen etal., Trends Microbiol. 2:198–201, 1994. Further, anti-MIF antibodieshave markedly increased survival in mice challenged with gram-positivebacteria that induces septic shock. Bernhagen et al., J. Mol. Med.76:151–161, 1998. Other studies have demonstrated the role of MIF intumor cell growth and that anti-sense inhibition of MIF leads toresistance to apoptotic stimuli. Takahashi et al., Mol. Med. 4:707–714,1998; Takahashi et al., Microbiol. Immunol. 43(1):61–67, 1999. Inaddition, the finding that MIF is a counterregulator of glucocorticoidaction indicates that methods of inhibiting MIF extracellularlocalization may allow for treatment of a variety of pathologicalconditions, including autoimmunity, inflammation, endotoxemia, and adultrespiratory distress syndrome, inflammatory bowel disease, otitis media,inflammatory joint disease and Crohn's disease. Bernhagen et al., J.Mol. Med. 76:151–161, 1998; Calandra et al., Nature 377:68–71, 1995;Donnelly et al., Nat. Med. 3:320–323, 1997. Because MIF is alsorecognized to be angiogenic, the inhibition of this cytokine may haveanti-angiogenic activity and particular utility in angiogenic diseasesthat include, but are not limited to, cancer, diabetic retinopathy,psoriasis, angiopathies, fertility, obesity and genetic diseases ofglucocorticoid dysfunction like Cushings and Addisons disease.

The inhibitors of MIF activity or export may be employed therapeuticallyand also utilized in conjunction with a targeting moiety that binds acell surface receptor specific to particular cells. Administration ofinhibitors or enhancers generally follows established protocols.Compositions of preferred embodiments may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments, the compositions describedherein may be administered as part of a sustained release implant.Within yet other embodiments, compositions of preferred embodiments maybe formulized as a lyophilizate, utilizing appropriate excipients thatprovide stability as a lyophilizate, and subsequent to rehydration.

In another embodiment, pharmaceutical compositions containing one ormore inhibitors of MIF are provided. For the purposes of administration,the compounds of preferred embodiments may be formulated aspharmaceutical compositions. Pharmaceutical compositions of preferredembodiments comprise one or more MIF inhibitors of preferred embodiments(i.e., a compound of structure (Ia) or (Ib)) and a pharmaceuticallyacceptable carrier and/or diluent. The inhibitor of MIF is present inthe composition in an amount which is effective to treat a particulardisorder, that is, in an amount sufficient to achieve decreased MIFlevels or activity, symptoms, and/or preferably with acceptable toxicityto the patient. Preferably, the pharmaceutical compositions of preferredembodiments may include an inhibitor of MIF in an amount from less thanabout 0.01 mg to more than about 1000 mg per dosage depending upon theroute of administration, preferably about 0.025, 0.05, 0.075, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg to about 65, 70, 75, 80, 85, 90,95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 375, 400, 425, 450,500, 600, 700, 800, or 900 mg, and more preferably from about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg to about 30, 35, 40, 45, 50, 55,or 60 mg. In certain embodiments, however, lower or higher dosages thanthose mentioned above may be preferred. Appropriate concentrations anddosages can be readily determined by one skilled in the art.

Pharmaceutically acceptable carriers and/or diluents are familiar tothose skilled in the art. For compositions formulated as liquidsolutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats, and other common additives. The compositions can also beformulated as pills, capsules, granules, or tablets that contain, inaddition to an inhibitor of MIF, diluents, dispersing and surface-activeagents, binders, and lubricants. One skilled in this art may furtherformulate the inhibitor of MIF in an appropriate manner, and inaccordance with accepted practices, such as those described inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

In addition, prodrugs are also included within the context of preferredembodiments. Prodrugs are any covalently bonded carriers that release acompound of structure (Ia) or (Ib) in vivo when such prodrug isadministered to a patient. Prodrugs are generally prepared by modifyingfunctional groups in a way such that the modification is cleaved, eitherby routine manipulation or in vivo, yielding the parent compound.

With regard to stereoisomers, the compounds of structures (Ia) and (Ib)may have chiral centers and may occur as racemates, racemic mixtures andas individual enantiomers or diastereomers. All such isomeric forms areincluded within preferred embodiments, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds ofstructures (Ia) and (Ib) may exist as polymorphs, which are included inpreferred embodiments. In addition, some of the compounds of structures(Ia) and (Ib) may also form solvates with water or other organicsolvents. Such solvates are similarly included within the scope of thepreferred embodiments.

In another embodiment, a method is provided for treating a variety ofdisorders or illnesses, including inflammatory diseases, arthritis,immune-related disorders, and the like. Such methods includeadministering of a compound of preferred embodiments to a warm-bloodedanimal in an amount sufficient to treat the disorder or illness. Suchmethods include systemic administration of an inhibitor of MIF ofpreferred embodiments, preferably in the form of a pharmaceuticalcomposition. As used herein, systemic administration includes oral andparenteral methods of administration. For oral administration, suitablepharmaceutical compositions of an inhibitor of MIF include powders,granules, pills, tablets, and capsules as well as liquids, syrups,suspensions, and emulsions. These compositions may also includeflavorants, preservatives, suspending, thickening and emulsifyingagents, and other pharmaceutically acceptable additives. For parentaladministration, the compounds of preferred embodiments can be preparedin aqueous injection solutions that may contain, in addition to theinhibitor of MIF activity and/or export, buffers, antioxidants,bacteriostats, and other additives commonly employed in such solutions.

As mentioned above, administration of a compound of preferredembodiments can be employed to treat a wide variety of disorders orillnesses. In particular, the compounds of preferred embodiments may beadministered to a warm-blooded animal for the treatment of inflammation,cancer, immune disorders, and the like.

MIF inhibiting compounds may be used in combination therapies with otherpharmaceutical compounds. In preferred embodiments, the MIF inhibitingcompound is present in combination with conventional drugs used to treatdiseases or conditions wherein MIF is pathogenic or wherein MIF plays apivotal or other role in the disease process. In particularly preferredembodiments, pharmaceutical compositions are provided comprising one ormore MIF inhibiting compounds, including, but not limited to compoundsof structures (1a) or (1b), in combination with one or more additionalpharmaceutical compounds, including, but not limited to drugs for thetreatment of various cancers, asthma or other respiratory diseases,sepsis, arthritis, inflammatory bowel disease (IBD), or otherinflammatory diseases, immune disorders, or other diseases or disorderswherein MIF is pathogenic.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with one or more nonsteroidalanti-inflammatory drugs (NSAIDs) or other pharmaceutical compounds fortreating arthritis or other inflammatory diseases. Preferred compoundsinclude, but are not limited to, celecoxib; rofecoxib; NSAIDS, forexample, aspirin, celecoxib, choline magnesium trisalicylate, diclofenacpotasium, diclofenac sodium, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, melenamicacid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam,rofecoxib, salsalate, sulindac, and tolmetin; and corticosteroids, forexample, cortisone, hydrocortisone, methylprednisolone, prednisone,prednisolone, betamethesone, beclomethasone dipropionate, budesonide,dexamethasone sodium phosphate, flunisolide, fluticasone propionate,triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide,betamethasone dipropionate, betamethasone valerate, desonide,desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide,clobetasol propionate, and dexamethasone.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with one or more beta stimulants,inhalation corticosteroids, antihistamines, hormones, or otherpharmaceutical compounds for treating asthma, acute respiratorydistress, or other respiratory diseases. Preferred compounds include,but are not limited to, beta stimulants, for example, commonlyprescribed bronchodilators; inhalation corticosteroids, for example,beclomethasone, fluticasone, triamcinolone, mometasone, and forms ofprednisone such as prednisone, prednisolone, and methylprednisolone;antihistamines, for example, azatadine, carbinoxamine/pseudoephedrine,cetirizine, cyproheptadine, dexchlorpheniramine, fexofenadine,loratadine, promethazine, tripelennamine, brompheniramine,cholopheniramine, clemastine, diphenhydramine; and hormones, forexample, epinephrine.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating IBD, such as azathioprine or corticosteroids, in apharmaceutical composition.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating cancer, such as paclitaxel, in a pharmaceutical composition.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with immunosuppresive compounds ina pharmaceutical composition. In particularly preferred embodiments, oneor more MIF inhibiting compounds are present in combination with one ormore drugs for treating an autoimmune disorder, for example, Lymedisease, Lupus (e.g., Systemic Lupus Erythematosus (SLE)), or AcquiredImmune Deficiency Syndrome (AIDS). Such drugs may include proteaseinhibitors, for example, indinavir, amprenavir, saquinavir, lopinavir,ritonavir, and nelfinavir; nucleoside reverse transcriptase inhibitors,for example, zidovudine, abacavir, lamivudine, idanosine, zalcitabine,and stavudine; nucleotide reverse transcriptase inhibitors, for example,tenofovir disoproxil fumarate; non nucleoside reverse transcriptaseinhibitors, for example, delavirdine, efavirenz, and nevirapine;biological response modifiers, for example, etanercept, infliximab, andother compounds that inhibit or interfere with tumor necrosing factor;antivirals, for example, amivudine and zidovudine.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating sepsis, such as steroids or anti-infective agents. Examples ofsteroids include corticosteroids, for example, cortisone,hydrocortisone, methylprednisolone, prednisone, prednisolone,betamethesone, beclomethasone dipropionate, budesonide, dexamethasonesodium phosphate, flunisolide, fluticasone propionate, triamcinoloneacetonide, betamethasone, fluocinolone, fluocinonide, betamethasonedipropionate, betamethasone valerate, desonide, desoximetasone,fluocinolone, triamcinolone, triamcinolone acetonide, clobetasolpropionate, and dexamethasone. Examples of anti-infective agents includeanthelmintics (mebendazole), antibiotics including aminoclycosides(gentamicin, neomycin, tobramycin), antifungal antibiotics (amphotericinb, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin,micatin, tolnaftate), cephalosporins (cefaclor, cefazolin, cefotaxime,ceftazidime, ceftriaxone, cefuroxime, cephalexin), beta-lactamantibiotics (cefotetan, meropenem), chloramphenicol, macrolides(azithromycin, clarithromycin, erythromycin), penicillins (penicillin Gsodium salt, amoxicillin, ampicillin, dicloxacillin, nafcillin,piperacillin, ticarcillin), tetracyclines (doxycycline, minocycline,tetracycline), bacitracin; clindamycin; colistimethate sodium; polymyxinb sulfate; vancomycin; antivirals including acyclovir, amantadine,didanosine, efavirenz, foscamet, ganciclovir, indinavir, lamivudine,nelfinavir, ritonavir, saquinavir, stavudine, valacyclovir,valganciclovir, zidovudine; quinolones (ciprofloxacin, levofloxacin);sulfonamides (sulfadiazine, sulfisoxazole); sulfones (dapsone);furazolidone; metronidazole; pentamidine; sulfanilamidum crystallinum;gatifloxacin; and sulfamethoxazole/trimethoprim.

In the treatment of certain diseases, it may be beneficial to treat thepatient with a MIF inhibitor in combination with an anesthetic, forexample, ethanol, bupivacaine, chloroprocaine, levobupivacaine,lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane,isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl,hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone,remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine,dibucaine, ethyl chloride, xylocaine, and phenazopyridine.

EXAMPLES

The inhibitors of MIF of preferred embodiments may be prepared by themethods described in Example 1. Example 2 presents an assay forscreening compounds of preferred embodiments for inhibition of activityor export.

Example 1

Synthesis of Representative Compounds

A solution of ethyl nitroacetate (6.70 ml, 60.1 mmol) inN,N-dimethylacetamide (35 ml) was treated with 95% NaH (1.52 g, 60.2mmol) in portions. After evolution of hydrogen ceased, the reactionmixture was heated at 80° C. for 15 minutes. A solution ofN-methylisatoic anhydride 1 (11.1 g, 62.5 mmol) in N,N-dimethylacetamide(65 ml) was added over a period of 15 minutes after which the reactionwas heated at 120° C. for 18 hours. The solvent was removed bydistillation, the residue dissolved in water, and acidified with 6 NHCl. The ensuing precipitate was collected and washed with water.Recrystallization of the remaining residue from CH₂Cl₂/ether gavequinolinone 2a (5.75 g, 43%) as a yellow solid.

A solution of diethyl malonate (18.8 ml, 111 mmol) inN,N-dimethylacetamide (35 ml) was treated with 95% NaH (2.80 g, 111mmol) in portions. After evolution of hydrogen ceased, the reactionmixture was heated at 80° C. for 15 minutes. A solution ofN-methylisatoic anhydride 1 (22.0 g, 124 mmol) in N,N-dimethylacetamide(140 ml) was added over a period of 15 minutes after which the reactionwas heated at 120° C. for 18 hours. The solvent was removed bydistillation, the residue dissolved in water, and acidified with 6 NHCl. The ensuing precipitate was collected and washed with water.Recrystallization of the remaining residue from CH₂Cl₂/ether gavequinoline 2b (11.7 g, 40%) as a white solid.

Alcohol 2 (6.2 g 2a; 11.1 g 2b) was dissolved in POCl₃ (70 mL for 2a;140 mL for 2b) and the solution was heated at 95° C. for three hours.POCl₃ was removed by distillation and the reaction mixture was pouredinto 500 mL of ice water. The aqueous solution was neutralized usingsaturated Na₂HCO₃, the precipitate was collected by filtration,redissolved in methylene chloride, dried over Na₂SO₄ and the solvent wasremoved in vacuo. The crude reaction product was purified byrecrystallization from ethyl ether affording 3.5 g of 3a (52%) or 7.8 g3b (65%).

N-BOC piperazine 4 (5.0 g; 27 mmol) was dissolved in pyridine (15 mL)and 20 mg of DMAP was added. Neat acid chloride was added to thesolution at 0–5° C. slowly over a period of 5 minutes. The resultingthick paste was stirred overnight (15 hours) at room temperature beforeit was poured on ice. The white crystalline precipitate was collected byfiltration, air dried and dried in vacuo to give the correspondingN-BOC-N-Acyl piperazine 5a (6.5 g; 83%) or 5b (4.8 g; 94%). The crudereaction product was immediately employed in the next step by dissolvingthe compound in 100 mL methylene chloride and adding neat TFA (10 mL).After 3 hours at room temperature, the solution was evaporated, theresidue dissolved in methylene chloride and washed with NaHCO₃(saturated). The aqueous layer was extracted 10 times with methylenechloride, the combined organic layer was dried over Na₂SO₄ and thesolvent was evaporated in vacuo affording compound 6a (4.5 g; 91%; 75%over two steps) or compound 6b (2.95 g; 70%; 66% over two steps), orcompound 6c, as is commercially available from Lancaster Synthesis(catalogue no. 18698).

To a solution of chloroquinolone 3 in toluene was added piperazine 6followed by 10 drops of pyridine before heated to 100° C. for 12 to 14hours. The mixture was cooled to room temperature, evaporated todryness, redissolved in methylene chloride and washed with brine. Theorganic solvent was dried over Na₂SO₄ and removed in vacuo.Chromatography (silica, CHCl₃: MeOH 85:15) afforded the CBX product 7,along with recovered starting material (30–40%). Product yields for thereaction are provided in Table 1.

TABLE 1 CBX N–Acyl Piperazine Chloroquinolone Piperazine QuinoloneAdduct 6a 1.9 g 3a 1.8 g 7a 0.9 g 28% 6a 1.6 g 3b 1.5 g 7b 0.9 g 34% 6b2.6 g 3a 2.5 g 7c 1.3 g 32% 6b 2.7 g 3b 2.4 g 7d 1.5 g 35% 6c 2.7 g 3a3.2 g 7e 2.8 g 55%

Analytical Data

7a. mp 167–159° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.95 (dd, 1H), 7.70 (dt,1H), 7.49 (d, 1H), 7.44 (d, 1H) 7.35 (m, 2H), 7.08 (m, 1H), 3.95 (bs,4H), 3.75 (s, 3H), 3.3 (t, 4H); HRMS (FAB) calculated for C₁₉H₁₉O₄N₄S399.1127, found 399.1117; Anal. Calculated for C₁₉H₁₈N₄O₄S: C, 57.28; H,4.55; N, 14.06; O, 16.06; S, 8.05 Found: C, 56.78; H, 4.50; N, 13.73.

7b. mp 215–217° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.85 (dd, 1H), 7.70 (m,1H), 7.44 (m, 5H), 7.42 (m, 1H), 3.74 (m, 4H), 3.28 (m, 4H); HRMS (FAB)calculated for C₂₁H₂₁N₄O₄ 393.1563, found 393.1540; Anal, Calculated forC₂₁H₂₀N₄O₄; C, 64.28; H, 5.14; N, 14.28; O, 16.31 Found; C, 64.84; H,5.16; N, 13.78.

7c. mp 183–185° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.95 (dd, 1H), 7.61 (m,1H), 7.48 (m, 5H), 7.42, (m, 1H) 7.32(m, 1H), 7.25 (M, 1H), 7.07 (M,1H), 4.44 (q, 2H) 3.95 (bs, 4H), 3.70 (s, 3H) 3.35 (bs, 4H), 1.44 (t,3H); HRMS (FAB) calculated for C₂₂H₂₄N₃O₄S, 426.1488 found 426.1487;Anal, Calculated for C₂₂H₂₃N₃O₄S: C, 62.10; H, 5.45; N, 9.88; O, 15.04;S, 7.54 Found: C, 62.08; H, 5.45; N, 9.77.

7d. mp 164–166° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.92 (dd, 1H), 7.60 (m,1H), 7.44 (m, 5H), 7.37 (m, 1H), 7.27 (m, 1H), 4.45 (q, 2H), 3.65 (bs,7H), 3.20 (bs, 4H), 1.45 (t, 3H); HRMS (FAB) calculated for C₂₄H₂₆NO₃,420.1923, found 420.1934; Anal. Calculated for C₂₄H₂₅N₃O₄; C, 69.72; H,6.01; N, 10.02; 0, 15.26 Found: C, 66.32; H, 6.01; N, 9.66.

7e. mp 189–191° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.96 (dd, 1H), 7.71 (dt,1H), 7,51 (bd, 1H), 7.44 (bd, 1H), 7.37 (bt, 1H), 7.09 (d, 1H), 6.51 (m,1H), 4.03 (bm, 4H), 3.74 (s, 3H), 3.33 (t, 4H); HRMS FAB) calculated forC₁₉H₁₉N₄O₅ 383.1355, found 383.1358; Anal. Calculated for C₁₉H₁₈N₄O₅; C,59.68; H, 4.74; N, 14.65 Found: C, 59.39, H, 4.79, N, 14.36.

Example 2

Macrophage Migration Assay

Macrophage migration is measured by using the agarose droplet assay andcapillary method as described by Harrington and Stastny et al., J.Immunol. 110(3):752–759, 1973. Briefly, macrophage-containing samplesare added to hematocrit tubes, 75 mm long with a 1.2 mm inner diameter.The tubes are heat sealed and centrifuged at 100×G for 3 minutes, cut atthe cell-fluid interface and imbedded in a drop of silicone grease inSykes-Moore culture chambers. The culture chambers contain either acontrol protein (BSA) or samples. Migration areas are determined after24 and 48 hours of incubation at 37° C. by tracing a projected image ofthe macrophage fans and measuring the areas of the migratin byplanimetry.

Alternatively, each well of a 96-well plate is pre-coated with onemicroliter of liquid 0.8% (w/v) Sea Plaque Agarose in water dispensedonto the middle of each well. The plate is then warmed gently on a lightbox until the agarose drops are just dry. Two microliters of macrophagecontaining 0.2% agarose suspensions of up to 25% (v/v) in media (with orwithout MIF or other controls), containing 0.2% (w/v) agarose (w/v) andheated to 37° C. is added to the precoated plate wells and cooled to 4°C. for 5 min. Each well is then filled with media and incubated at 37°C. under 5% CO₂—95% air for 48 hr. Migration from the agarose dropletsis measured at 24 and 48 hr by determining the distance from the edge ofthe droplet to the periphery of migration.

Migration Assay

Monocyte migration inhibitory activities of recombinant murine and humanwild-type and murine mutant MIF are analyzed by use of human peripheralblood mononuclear cells or T-cell depleted mononuclear cells in amodified Boyden chamber format. Calcein AM-labeled monocytes aresuspended at 2.5 to 5×10⁶/mL in RPMI 1640 medium, with L-glutamine(without phenol red) and 0.1 mg/mL human serum albumin or bovine serumalbumin. An aliquot (200 μL) of cell suspension is added to wells of aU-bottom 96-well culture plate (Costar, Cambridge, Mass.) prewarmed to37° C. MIF in RPMI 1640 is added to the cell suspension to yield finalconcentrations of 1, 10, 100, and 1000 ng/mL. The culture plate isplaced into the chamber of a temperature-controlled plate reader, mixedfor 30 s, and incubated at 37° C. for 10–20 min. During the incubation,28 μL of prewarmed human monocyte chemotactic protein 1 (MCP-1; PeproTech., Inc., Rocky Hill, NJ) at 10 or 25 ng/mL or RPMI 1640 with 0.1mg/mL HSA is added to the bottom well of a ChemoTX plate (Neuro ProbeInc., Gaithersburg, Md.; 3 mm well diameter, 5 μM filter pore size). Thefilter plate is carefully added to the base plate. Treated cellsuspensions are removed from the incubator and 30 μL is added to eachwell of the filter plate. The assembled plate is incubated for 90 min.at 37° C. in a humidified chamber with 5% CO₂. Following incubation, thecell suspension is aspirated from the surface of the filter and thefilter is subsequently removed from the base plate and washed threetimes by adding 50 μL of 1×HBSS⁻ to each filter segment. Between washes,a squeegee (NeuroProbe) is employed to remove residual HBSS⁻. The filteris air-dried and then read directly in the fluorescent plate reader,with excitation at 485 nm and emission at 535 nm. Chemotactic or randommigration indices are defined as average filter-bound fluorescence for agiven set of wells divided by average fluorescence of filters in wellscontaining neither MCP-1 nor MIF. Titration of fluorescently labeledcells revealed that levels of fluorescence detected in this assay have alinear relationship to cell number (not shown).

Example 3

Tautomerase Assay

The tautomerization reaction is carried out essentially as described byRosengren et al., Mol. Med. 2(1):143–149, 1996. D-dopachrome conversionto 5,6-dihydroxyindole-2-carboxylic acid is assessed. 1 ml samplecuvettes containing 0.42 mM substrate and 1.4 μg of MIF in a samplesolution containing 0.1 mM EDTA and 10 mM sodium phosphate buffer, pH6.0 are prepared and the rate of decrease in iminochrome absorbance isfollowed at 475 nm. L-dopachrome is employed as a control. In addition,the reaction products can be followed using an HPLC, utilizing a mobilephase including 20 mM KH₂PO₄ buffer (pH 4.0) and 15% methanol with aflow rate of 1.2 m/min. Fluorimetric detection is followed at 295/345nm.

Alternatively, the tautomerization reaction utilizing phenylpyruvate or(p-hydroxyphenyl)pyruvate is carried out essentially as described byJohnson et al., Biochem. 38:16024–16033, 1999. In this version,ketonization of phenylpyruvate is monitored at 288 nm (ε=17300 M⁻¹ cm⁻¹)and the ketonization of (p-hydroxyphenyl)pyruvate is monitored at 300 nm(ε=21600 M⁻¹ cm⁻¹). The assay mixture contains 50 mM Na₂HPO₄ buffer (1mL, pH 6.5) and an aliquot of a solution of MIF sufficiently dilute(0.5–1.0 μL of a 2.3 mg/mL solution, final concentration of 93–186 nM)to yield an initial liner rate. The assay is initiated by the additionof a small quantity (1–3.3 μL) of either phenylpyruvate or(p-hydroxyphenyl)pyruvate from stock solutions made up in ethanol. Thecrystalline forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate existexclusively as the enol isomers (Larsen et al., Acta Chem. Scand. B28:92–96, 1974). The concentration of substrate may range from 10 to 150M, with no significant inhibition of MIF activity by ethanol observed atless than 0.5% v/v.

Example 4

Immunoprecipitation and Western Blot Analysis

Cell culture experiments were designed to characterize the activity ofcandidate compounds, MIF expression, trafficking, and export. Cell andconditioned medium fractions are prepared for immunoprecipitationessentially as described previously (Florkiewicz et al., Growth Factors4:265–275, 1991; Florkiewicz et al., Ann. N.Y. Acad. Sci. 638:109–126)except that 400 μl of lysis buffer (1% NP-40, 0.5% deoxycholate, 20 mMTris pH 7.5, 5 mM EDTA, 2 mM EGTA, 0.01 mM phenylmethylsufonyl fluoride,10 ng/ml aprotinin, 10 ng/ml leupeptin, 10 ng/ml peptstatin) is added tothe medium fraction after clarification by centrifugation in a microfugefor 15 minutes. Cell or medium fractions are incubated with monoclonalor polyclonal antibodies to MIF and GammaBind™ G Sepharose® (PharmaciaLKB Biotechnology, Uppsala, Sweden) was added for an additional 30minutes incubation. Immune complexes are sedimented by microfugecentrifugation, washed three times with lysis buffer, and four timeswith ice cold immunoprecipitation wash buffer (0.15M NaCl, 0,01 MNa-phosphate pH 7.2, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecylsulfate). Immune complexes are dissociated directly in SDS gel samplebuffer 125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.004% bromphenolblue, 2 mM EGTA, and separated by 12% SDS-PAGE. The gel is processed forfluorography, dried, and exposed to X-ray film at −70° C. When neomycinphosphotransferase is immunoprecipitated, a rabbit anti-NPT antibody(5Prime-3Prime, Boulder, Colo.) was employed.

For Western blot analysis, proteins are transferred from the 12%SDS-PAGE gel to a nitrocellulose membrane (pore size 0.45 μm in coldbuffer containing 25 mM3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropane-sulfonic acid,pH 9.5, 20% methanol for 90 minutes at 0.4 amps. For Western blottinganalysis, of cell conditioned media, the media was centrifuged (10minutes at 800 g) and the supernatants concentrated 10-fold by membranefiltration (10 kDa cut-off, Centricon-10 Amicon). Samples were thenresolved on 16% SDS Tris-glycin Gel (Novex, San Diego, Calif.) underreducing condition and transferred onto nitrocellulose membrane (Novex)at 20V for 3 hours. Membrane was incubated with rabbit polyclonalanti-rat antibodies (1:1000) (Torrey Pines Biolab, San Diego, Calif.),and then with horseradish peroxidase-conjugate (1:1000)(Pierce,Rockford, Ill.). MIF was visualized by development withchloronaphtnol/H₂O₂. Recombinant MIF (2 ng, purchased from R&D systems,Minneapolis) was electrophoresed and transferred as a standard.Membranes are blocked in 10 mM Tris, pH 7.5, 150 mM NaC1, 5 mM NaN₃,0.35% polyoxyethylene-sorbitan monolaurate, and 5% nonfat dry milk(Carnation Co., Los Angeles, Calif.) for 1 hr at room temperature.Membranes are incubated with a monoclonal antibody (Catalog NumberMAB289, purchased from R&D Systems, Minneapolis, Minn.) or polyclonal(goat polyclonal serum, R&D Systems cat#AF-289-PB). Followingincubation, membranes are washed at room temperature with 10 changes ofbuffer containing 150 mM NaCl, 500 mM sodium phosphate pH 7.4, 5 mMNaN₃, and 0.05% polyoxyethylene-sorbitan monolaurate. When usingmonoclonal antibodies, membranes are then incubated in blocking buffercontaining 1 μg/ml rabbit anti-mouse IgG (H+ L, affinipure, JacksonImmuno Research Laboratories, West Grove, Pa.) for 30 minutes at roomtemperature. For polyclonal probing, incubation employed rabbitanti-goat (Sigma, Catalog Number G5518). Membranes are subsequentlywashed in 1 L of buffer described above, and incubated for 1 hr in 100ml of blocking buffer containing 15 μCi ¹²⁵I-protein A (ICNBiochemicals, Costa Mesa, CA), and washed with 1 L of buffer. Theradiosignal is visualized by autoradiography.

In one experiment, overnight conditioned media was collected from LPS(10 μg/ml) treated THP-1 cells also treated with varying amounts ofcandidate compounds, such as compound 7e and screened byimmunoprecipitation with monoclonal or polyclonal antibodies to detectMIF binding. As demonstrated in FIG. 1, conditioned media showed asignificant loss of detectable MIF using the monoclonal antibody in thepresence of 10 μM of compound 7e that was not observed with thepolyclonal antibody. This response mirrors the effects of compound 7e onMIF enzyme activity. Accordingly, this experiment demonstrates thatmonoclonal reactivity can act as a surrogate marker for enzymaticactivity.

In another experiment (FIG. 2), varying concentrations of five differentinhibitor analogs were added to LPS stimulated THP-1 cells and allowedto incubate overnight. The following day the amount of immunoreactiveMIF detected was evaluated by ELISA. Compound 7e inhibited the abilityof the antibody to recognize MIF in a dose dependent fashion with anED50 of 2 μM, similar to the response obtained with analogs compound 7band compound 7d. In contrast, analog compound 7a and compound 7c werealmost 100 times more active.

In a further experiment (FIG. 3), the ability of compound 7e to decreasethe immunoreactivity of MIF produced by THP-1 cells was determined.THP-1 cells were treated with 10 μg/ml of LPS and 10 μM of compound 7ewas added at various times post-LPS stimulation and immunoreactivitymonitored with an anti-MIF monoclonal. As shown, following addition ofcompound 7e immunoreactivity is rapidly lost. Thus, this experimentmeasures the activity of compounds or buffer alone controls on MIFdetection when the compounds are initially added at various times tocell cultures and then the corresponding conditioned media samples areprocessed in a time dependent fashion thereafter.

In the previous experiment (FIG. 3), the ability of compound 7e tomodulate antibody binding to MIF protein was analyzed in the presence ofLPS-stimulated THP1 cells. However, in the experiment shown in FIG. 4,the ability of compound 7e to modulate antibody recognition of MIF wasexamined using pre-conditioned media, in the absence of live cells. Inthis experiment, LPS was added to THP1 cells in culture as describeabove. Six hours later, the conditioned media was removed, clarified ofcell debris and the amount of MIF determined to be 22 ng/ml. Thispre-conditioned media was then divided into two groups. Both groups wereincubated at 37° C. for varying periods of time before compound 7e orbuffer alone (control) was added for an additional 30 minutes ofincubation at 37° C. The level of detectable MIF was then determined byELISA using the monoclonal anti-MIF antibody for detection. The rapidloss of MIF specific ELISA signal is dependent upon the presence ofcompound 7e. Control levels of MIF do not change. Accordingly, thisexperiment demonstrates that compound 7e interacts with MIF, and blocksthe antibody's ability to subsequently interact with MIF, even in theabsence of cells. As this interaction takes place at the catalytic site,or constrains catalytic activity, the loss of immunoreactivitycorrelates with lost enzymatic activity and/or MIF associatedactivities.

Example 5

Extracellular Localization Assay

In order to further assess in vitro activity of compound 7e to modulateMIF export, mouse macrophage RAW 264.7 cells (American Type CultureCollection, Manassas, Va.) were selected.

Raw 264.7 macrophage (3×10⁶ cells per well) were plated in 12-welltissue culture plates (Costar) and cultured in RPMI/1% heat-inactivatedfetal bovine serum (FBS) (Hyclone Laboratories, Logan, Utah). Afterthree hours of incubation at 37° C. in a humidified atmosphere with 5%CO₂, nonadherent cells were removed and wells were washed twice withRPMI/1% FBS. Cells were then incubated for 24 hours with LPS (0111:B4)or TSST-1 (Toxin Technology, Sarasota, FL), which were approximately 95%pure and resuspended in pyrogen-free water, at a concentration rangingfrom 1 pg/ml to 1000 ng/ml (for the dose response experiment). Fortime-course experiments, conditioned media of parallel cultures wereremoved at 0.5, 1, 2, 4, 8 and 24 hours intervals after stimulation with1 ng/ml TSST-1 or LPS. For the inhibition studies, RAW 264.7 cells(3×10⁶ cells per well) were incubated for 24 hours with 1 ng/ml of LPS(0111:B4) or 1 ng/ml of TSST-1 in the presence of 0.01 μM to 10 μMcompound 7e or buffer (as control). The MIF in cell-conditioned mediawas concentrated on filters and the MIF remaining in the samples wasanalyzed by Western blotting and MIF band densities were also measuredby Stratagene Eagle Eye™.

RAW cells can be induced to express MIF by addition either 1 ng/mlTSST-1 or LPS and cultured for 24 hours. MIF in conditioned media wasmeasured as described above. As demonstrated by FIG. 5, compound 7ereduced immunodetectable MIF levels in conditioned media in aconcentration dependent manner with an IC₅₀ of approximately at 0.04 μM,as compared to cells incubated with buffer only. The level of MIFdetected in the presence of compound 7e following TSST-1 stimulation ofRAW cells is illustrated in FIG. 6, with an IC₅₀ of approximately 0.3 μMas compared to cells incubated with buffer only.

Example 6

Cell Culture, Transfection, and Metabolic Labeling

Target cells obtained from the American Type Culture Collection (ATCCNo. CRL 1650) are cultured overnight in a 48-well plate in DMEMsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodiumpyruvate, 100 nM nonessential amino acids, and 50 μg/ml gentamycin. Thetarget cells are then transfected with 2 μg/ml of CsCl-purified plasmidDNA in transfection buffer (140 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 0.5 mMMgCl₂, 0.9 mM Na₂HPO₄, 25 mM Tris, pH 7.4. To each well, 300 μl of theDNA in transfection buffer is added. Cells are incubated for 30 minutesat 37° C., and the buffer is aspirated. Warn medium supplemented with100 μm chloroquine is added for 1.5 hr. This medium is removed and thecells are washed twice with complete medium. Cells are then incubatedfor 40–48 hr. The plasmid of interest is co-transfected with pMAMneo(Clontech, Palo Alto, Calif.), which contains the selectable markerneomycin phosphotransferase. When 2 μg of the plasmid of interest areco-transfected with 10 μg of pMAMneo, greater than 70% of transfectedcells express both MIF and neo, as determined by immunofluorescencemicroscopy.

For immunoprecipitation assays the target cells are metabolicallypulse-labeled for 15 minutes with 100 μCi of ³⁵S-methionine and³⁵S-cysteine (Trans ³⁵S-label, ICN Biomedicals, Irvine, Calif.) in 1 mlof methionine and cysteine free DMEM. Following labeling, the cellmonolayers are washed once with DMEM supplemented with excess (10 mM)unlabeled methionine and cysteine for 1–2 minutes. Cells are thencultured in 2 ml of this medium for the indicated lengths of time andthe cell supernatants are immunoprecipitated for the presence ofleaderless protein. For the indicated cultures, chase medium issupplemented with modulator at the indicated concentrations.

Alternatively, for analysis by ELISA, the target cells are washed oncewith 250 μl of 0.1 M sodium carbonate, pH 11.4, for 1 to 2 minutes andimmediately aspirated. A high salt solution may alternatively bepreferred. The cells are washed with media containing 0.5% FBS plus 25μg/ml heparin and then the cells are incubated in this same medium forthe indicated lengths of time. For indicated cultures, chase medium issupplemented with a modulator. For cells transfected with vectorencoding a protein containing a leader sequence, such as hCG-α or anyother non-heparin binding protein, the carbonate wash and heparincontaining medium may be omitted.

Example 7

High Throughput Screening Assay for MIF Inhibitors

The high throughput screening assay for MIF inhibitors is performed in a96-well format using MIF produced by THP-1 cells and is performed asfollows. MIF assays are performed by ELISA as indicated above. THP-1cells are resuspended to approx. 5×10⁶ cells/ml in RPMI mediumcontaining 20 μg/ml of bacterial LPS and the cells incubated for 18–20hours. Subsequently cell supernatant is collected and incubated withputative inhibitors. Briefly, a 96-well plate (Costar Number 3590) ELISAplate is coated with a MIF monoclonal antibody (R&D Systems CatalogNumber MAB289) at a concentration of 4 μg/ml for two hours at 37° C.Undiluted culture supernate is added to the ELISA plate for a two-hourincubation at room temperature. The wells are then washed, abiotinylated MIF polyclonal antibody (R&D Systems #AF-289-PB) is addedfollowed by Streptavidin-HRP and a chromogenic substrate. The amount ofMIF is calculated by interpolation from an MIF standard curve.

Example 8

HPLC Analysis of Candidate Inhibitors in Serum

Prior to evaluating the affects of any small molecule in vivo, it isdesirable to be able to detect, in a quantitative fashion, the compoundin a body fluid such as blood. An analytical method was established tofirst reproducibly detect test compounds, such as MIF inhibitorsincluding compound 7e, and then measure its concentration in biologicalfluid.

RP-HPLC was performed with a Hewlett-Packard Model HP-1100 unit usingSymmetry Shield RP-8 (4.6×75 mm id, Waters, Milford, MA). The mobilephase was an isocratic solution of 35% Acetonitrile/water containing0.1% trifluroacetic acid. Absorbance was monitored at 235 nm. To measurethe amount of test compound in serum, the sample serum proteins werefirst separated using 50% Acetonitrile (4° C. overnight) followed bycentrifugation at 14000 rpm for 30 minutes. The supernatant was thenanalyzed by the RP-HPLC and the compound concentration calculated basedon a calibration curve of known standard. According to this procedure,reverse phase HPLC was employed to detect compound 7e in a linear rangeof 1.5–800 ng (R2=1) using spiked test samples (not shown). When theabove analytical technique is applied to blood serum from animalsreceiving compound 7e (0.4 mg/20 gram mouse), circulating concentrationsof compound 7e are quantitatively measured.

With the development of the above methods to quantify compound 7e, it ispossible to evaluate the efficacy of different routes of compoundadministration and to characterize bioactivity. To test time dependentserum bioavailability, animals were treated with compound 7e byintraperitoneal injection (i.p.) (FIG. 7A), and orally by gavage (FIG.7B).

Example 9

In Vivo Inhibition of MIF

The purpose for the following in vivo experiments was to confirm initialin vitro assay results using compound 7e to inhibit MIF. LPS-inducedtoxicity appears to be related to an overproduction of MIF as well asTNF-α and IL-1β. Since animals can be protected from endotoxin shock byneutralizing or inhibiting these inflammation mediators. The presentmodel was chosen because it provides reproducible and rapid lethalmodels of sepsis and septic shock.

Doses of lipopolysacchraride (LPS) were made fresh prior to eachexperiment. LPS (Escherichia Coli 0111:B4, Sigma) was reconstituted byadding 0.5% TEA (1 ml USP water+5 ml Triethylamine (Pierce)) to a vialof 5 mg endotoxin. Once reconstituted, the solution was incubated at 37°C. for 30 minutes. Subsequently, the solution was sonicated in a 56–60°C. bath sonicator for 30 seconds 3 times. Following sonication themixture was vortexed for 3 minutes continuously. The stock solution ofLPS was then ready for use.

Detection of IL-1β and TNF-α and MIF in Blood

Ten 10-week-old (20±2 gram) female BALB/c mice (Charles RiverLaboratories, Kingston, NY) were housed in a group of 5 per cage withfree access to food and water and were acclimatized for at least oneweek prior to experimentation. On the day of experiment, mice wereweighed and randomly distributed into group of 10 animals of equal meanbody weight. Mice were injected i.p. with 200 μL of formulated compound7e or buffer alone immediately before the i.p. injection of LPS(Escherichia coli 0111:B4, 10 mg/kg or 5 mg/kg body weight) andβ-D-galactosamine (50 mg/kg body weight). Each dose of LPS (0.2 ml for20 gram mouse) was administered intraperitoneally and mixed with a finalconcentration of β-D-galactosamine of 50 mg per ml. Following collectionof blood specimens taken from cardiac puncture, the animal wassacrificed. Typical collections were performed at 4 hours post LPStreatment. The serum was separated in a serum separator (Microtainer®Becton Dickinson, Minneapolis, NJ) according to the manufacturer'sprotocol. Mouse serum Il-1β and TNF-α were measured by ELISA using a“mouse IL 1β immunoassay or mouse TNF-α immunoassay” kits (R&D SystemMinneapolis, Minn.) following manufacturer's direction. Serum MIFconcentrations in mouse serum were quantified by a sandwich ELISA(ChemiKine MIF Kit, Chemicon, San Diego, Calif.). Samples were analyzedin duplicate, and results were averaged.

Murine LPS Model

Ten 8 to 10 week-old (20±2 gram) female BALB/c mice were housed andacclimatized as described above. On the day of the experiments, the micewere weighed and randomly distributed into group of 5 animals of equalmean body weight. Mice were injected with 200 μl of formulated compound7e or its Buffer (average 20 mg/kg compound) following i.p. injection ofLPS (E. Coli 055B5, Sigma) (40, 10, 5, 2 or 0.5 mg/kg body weight) and50 mg/kg of β-D-galactosamine. Mice were observed every two hours duringthe first 18 hours and twice a day for seven days. For these studiesKaplan-Meier estimation methods were employed to assess animal survival.

For all in vivo studies, standard statistical comparisons amongtreatment groups were performed using the Fisher test for categoricaldata and the Mantel-Cox test for continuous variables. To determine iflevels of serum IL-1 correlated to serum MIF, a Fisher's test wasapplied. The analyses were performed using Stat View 5.0 Software(Abacus Concepts, Berkeley, CA). All reported p values that weretwo-sided and of a value less than 0.05 were considered to indicatestatistical significance.

An initial control experiment was conducted to determine the base linelevels of endogenous MIF in the murine model system (female Balb/cmice), and further to determine the rate and extent of increase inendogenous MIF following treatment with LPS (10 mg/kg). Female Balb/cmice were treated with LPS (Sigma 0111:B1) admixed with 50 mg/kgβ-D-galactosamine. The level of MIF in serum was measured by HPLC asdescribed above at 0, 2, 5 and 6 hours following LPS/galactosaminetreatment. At the initiation of this representative experiment, thebaseline level of endogenous MIF was approximately 45 ng/ml. However,over the course of this six-hour experiment there was a time dependentincrease in the level of MIF detected in collected serum samples. Whenmice were treated with compound 7e (formulated in 50% aqueous solution)and 10 mg/kg of LPS there was a significant decrease in the level ofcirculating MIF (p=0.05) that can be detected. In the experiment shownin FIG. 9A, BALB/c (n=20) mice were injected i.p. with 20 mg/kg bodyweight of compound 7e at time of LPS administration. Blood samples werecollected 5.5 hours later. The results demonstrate that animals treatedwith the inhibitor have a decreased ability to respond to LPS andlowered MIF levels are detected. In a further study, in which mice wereadministered with half the LPS dosage (5 mg/kg), serum MIF wasdetermined four hours following treatment. This data reveal a highlystatistically significant (p=0.0003) 60% decrease in MIF (FIG. 9B). In afurther experiment, both MIF and IL-1β were measured in mouse serum viaELISA. As shown in FIG. 10, there is a direct and highly significantcorrelation between the two. This correlation was also observed betweenMIF and TNF-α (data not shown). In a similar experiment, reductions inserum IL-1β level and serum TNF-α level were observed followingadministration of 20 mg/kg compound 7e (FIG. 11).

Studies of experimental toxic shock induced by LPS have revealed acentral role for MIF and TNF-α. The fact that LPS stimulatesmacrophage-like cells to produce MIF, that in turn induce TNF-αsecretion by macrophage like cells suggests a potential role for MIF inthe pathogenesis of LPS. To test if compound 7e can prevent LPS shock, amodel of lethal LPS mediated shock in BALB/c mice sensitized withβ-D-galactosamine was employed. Treatment with compound 7e at the timeof injection of a lethal dose of LPS (2, 5 and 10 mg/kg) increasedsurvival from 6% to 47% (p=0.0004) (FIG. 12). The effects are modulatedby the concentration of LPS employed, demonstrating that when using ahigher concentration of LPS, the effect compound 7e is saturable andhence specific. Table 2 is a summary of several survival experiments(total of 210 mice), indicating that compound 7e protects mice from LPSinduced toxic shock in a concentration dependent fashion. FIG. 13 alsodepicts this data in graphical form with 25% survival time on the leftaxis.

TABLE 2 LPS Dosage 75% Animal Death (hours) (mg/kg) Vehicle Compound 7e40 10.2 11.6 10 9.9 18.0 5 10.0 32.0 2 10.2 >100 0.5 22.0 >100 0.1 >100>100

MIF Overcomes the Effects of Compound 7e

Exogenous recombinant human MIF when administered with compound 7e, canreverse the beneficial effects of the compound, supporting thehypothesis that compound 7e acts to increase animal resistance to LPS bymodulating MIF levels in mice serum. In this example, mice were treatedwith the standard LPS protocol except that in addition to 1 mg/kg LPSand 20 mg/kg of the inhibitor compound 7e, some animals also received300 μg/kg human recombinant MIF. At 12 hours, significantly more(p<0.01) mice survive the LPS with compound 7e, but this survival isneutralized by the administration of MIF (data not shown).

Example 10

MIF Inhibitor in a Collagen Induced Arthritis Model

Twenty DBA/1LacJ mice, age 10–12 weeks, were immunized on day 0 at baseof the tail with bovine collagen type II (CII 100 μg) emulsified inFreunds complete adjuvant (FCA; GibcoBRL). On day 7, a second dose ofcollagen was administrated via the same route (emulsified in Freundsincomplete adjuvant). On Day 14 mice were injected subcutaneously with100 mg of LPS (055:B5). On day 70 mice were injected 40 μg LPS (0111:B4)intraperitoneally. Groups were divided according paw thickness, whichwas measured by a caliper, after randomization, to create a balancedstarting group. Compound in buffer was given to mice on days 71, 72, 73,and 74 (total eight doses at 0.4 mg/dose, approximately 20 mg/kg of bodyweight). Mice were then examined on day 74 by two observers for pawthickness. FIG. 14 sets forth the experimental timeline. In thisexperiment, subsided mice (decline of full-blown arthritis) were treatedwith a final i.p. injection of LPS on day 70 to stimulate cytokineproduction as well as acute inflammation. FIG. 15 demonstrates thatcompound 7e treated mice develop mildly reduced edema of the paw (1.87mm) compared with vehicle only treated controls (1.99 mm), p<0.05. Inthe late time point, the animals in the treated group did not reach afull-blown expression of collagen induced arthritis as compared to itscontrol (data not shown).

In another experiment, fifteen DBA/IJ mice, age 10–12 weeks wereimmunized on day 0 at the base of the tail with bovine collagen type II(CII 100 μg), emulsified in Freunds complete adjuvant (FCA; GibcoBRL).On day 21, a second dose of collagen was administered via the sameroute, emulsified in Freunds incomplete adjuvant. On day 28 the micewere injected subcutaneously with 100 μg of LPS (055:B5). On day 71 themice were injected i.p. with 40 μg LPS (0111:B4). Groups and treatmentprotocol were the same as described as above. On day 74 blood sampleswere collected and cytokines were measured. FIG. 16 indicates thatcompound 7e reduced serum MIF levels as compared to untreated CIAsamples. An even more significant inhibition of serum TNF-α levels wasdetected.

Example 11

The following inhibitors of MIF were prepared by the methods describedin Example 1. Each of these MIF inhibitors belongs to the class ofcompounds of structure (1a) described above, and incorporates thefollowing moiety:

Results of tautomerase assays indicated that each of the MIF inhibitorcompounds exhibited significant inhibition of MIF activity.

Example 12

Results of tautomerase assays indicated that the following compounds ofExample 11 exhibited particularly high levels of inhibition of MIFactivity.

TABLE 3 EC50 Tautomerase Rank Compound (mM) THP-1/MIF Structure 1 340.01 <0.008

2 126 0.01 <0.008

3 164 0.01

4 178 0.013 —

5 51 0.016 <0.008

6 50 0.018 <0.008

7 177 0.019 —

8 40 0.02 <0.008

9 202 0.023 —

10 28 0.029 —

11 57 0.034 0.015 (0.011–0.019)

12 49 0.04 0.084 (0.015–0.47) 

13 147 0.04 —

14 163 0.04 —

15 176 0.045 —

16 92 0.049 —

17 200 0.054 —

18 107 0.063 <0.008

19 26 0.07 —

20 105 0.075 —

21 16 0.08 0.03 (0.02–0.04)

22 27 0.08 —

23 29 0.08 —

Example 13

The following inhibitors of MIF were prepared. Each of these MIFinhibitors belongs to the class of compounds having structure (Ib)described above:

wherein R₁, R₂, R₃, R₄, X, Y, Z and n are as defined for structure (Ib)above. Results of tautomerase assays indicated that each of the MIFinhibitor compounds exhibited significant inhibition of MIF activity.

Example 14

Inhibitors of MIF of certain embodiments may be prepared according tothe following reaction schemes, Scheme 5 and Scheme 6. Each of these MIFinhibitors belongs to the class of compounds of structure (1a) describedabove.

Reaction Scheme 5

In this scheme, isatoic anhydride is reacted with diethyl malonate in asolution of NaH in N, N-dimethylacetamide. The resulting intermediate(referred to as “1M00”) is then chlorinated by reaction with POCl₃ toyield an intermediate (referred to as “1M00(Cl2)”). 1M00(Cl2) is thenreacted with NH₄OAC in acetic acid to yield an intermediate (referred toas “1M00(Cl)”). 1M00(Cl) is then reacted with an N-acyl piperazine inDMF. The acyl group of the piperazine compound includes as a substituent(referred to as “R3”) either a furanyl group or a thienyl group, asdepicted in Scheme 5, or other groups, as enumerated in subsequentexamples. The resulting intermediate is then reacted with a halogentcompound. The subsituent bound to the halogen atom (referred to as“R4”), may include various groups, as enumerated in subsequent examples.The resulting compound is of the structure (1a) described above. Thesteps in this reaction scheme are described in detail below. Compoundsprepared according to Scheme 5 are referred to below by referencenumbers containing an “M” and incorporate a —COOEt moiety.

Reaction Scheme 6

In this scheme, 4-Hydroxy-2(1H)-quinolone is reacted with a mixture ofnitric and acetic acids. The resulting intermediate is then chlorinatedby reaction with POCl₃ to yield another imtermediate. That intermediateis then reacted with an N-acyl piperazine in DMF. The acyl group of thepiperazine compound includes as a substituent an R3 group as referred toin the description of Scheme 5. The resulting intermediate is thenreacted with a halogen compound including as a substituent an R4 groupas referred to in the description of Scheme 5. The resulting compound isof the structure (1a) described above. The steps in this reaction schemeare described in detail below. Compounds prepared according to Scheme 6are referred to below by reference numbers containing an “N” andincorporate a —NO₂ moiety.

Various inhibitors of MIF belonging to the class of compounds having thestructure I(a) were prepared according to Scheme 5 or Scheme 6. Table 4provides a list of reference numbers for the compounds prepared. Thedesignation “1M1##” indicates that the compound was prepared by Scheme5, and incorporates a —COOEt moiety and a furan moiety as R3. Thedesignation “1M2##” indicates that the compound was prepared by Scheme5, and incorporates a —COOEt moiety and a thiophen moiety as R3. Thedesignation “1N1##” indicates that the compound was prepared by Scheme6, and incorporates a —NO₂ moiety and a furanyl moiety as R3. Thedesignation “1N2##” indicates that the compound was prepared by Scheme6, and incorporates a —NO₂ moiety and a thiophen moiety as R3. The twodigits at the end of the designation identify the compound's R4 group.

TABLE 4 M (COOEt) N (NO₂) Halogen - R4 1M1 (Furan) 1M2 (Thiophen) 1N1(Furan) 1N2 (Thiophen) 06

1M106 1M206 1N106 1N206 07

1M107 1M207 1N107 1N207 08

1M108 1M208 1N108 1N208 09

— — — — 10

1M110 1M210 1N110 1N210 11

1M111(1) 1M211(1) 1N111(1) 1N211(1) 12

1M112 1M212 1N112 1N212 13

1M113 1M213 1N113 1N213 14

1M114 1M214 1N114 1N214 15

1M115 1M215 1N115 1N215 16

1M116 1M216 1N116 1N216 17

1M117 1M217 1N117 1N217 18

1M118 1M218 1N118 1N218 19

1M119 1M219 1N119*HCl 1N219 20

1M120 1M220 1N120 1N220 22

1M122 1M222 1N122 1N222

The halogenated R4 group “09” is disclosed in MARCH'S ADVANCED ORGANICCHEMISTRY, Reactions, Mechanisms, and Structure, 5^(th) Ed., Michael B.Smith and Jerry March, Eds., A Wiley-Interscience Publication, JohnWiley & Sons, Inc., p. 437 (2001). Slighly different reaction schemes,Schemes 7 and 8, are used to prepare inhibitors of MIF incorporatingthis moiety.

Various inhibitors of MIF belonging to the class of compounds having thestructure I(a) were prepared according to Scheme 9 or Scheme 10.

Table 5 provides a list of reference numbers for the compounds prepared.The designation “1M##1” indicates that the compound was prepared byScheme 9, and incorporates a —COOEt moiety. The designation “1M##2”indicates that the compound was prepared by Scheme 9, and incorporates a—NO₂ moiety. The designation “1N##1” indicates that the compound wasprepared by Scheme 10, and incorporates a —COOEt moiety. The designation“1N##2” indicates that the compound was prepared by Scheme 10, andincorporates a —NO₂ moiety. The two digits following the letter M or Ncorrespond to the number identifying the R3 moiety.

TABLE 5 # R3 R3 1M##1 (COOEt) 1N##1 (NO2) 1 07

1M071*HCl 1N071*HCl 2 08

1M081 1N081 3 09

1M091 1N091 4 10

1M101 1N101 5 11

1M111(2) 1N111(2) 6 12

1M121 1N121 7 13

1M131 1N131 8 14

1M141 1N141 9 15

1M151*HCl 1N151*HCl 10 16

1M161 1N161 11 17

1M171 1N171 12 18

1M181 1N181 13 19

1M191*HCl 1N191 14 20

1M201 1N201 15 21

1M211(2)*HCl 1N211(2)*HCl 16 22

1M221 1N221 17 23

1M231 1N231 18 24

1M241 1N241 19 27

1M271 1N271 20 28

1M281 1N281 21 29

1M291 1N291 22 30

1M301 1N301 23 31

1M311 1N311 24 32

1M321 1N321 25 33

1M331 1N331 26 34

1M341 1N341 27 35

1M351 1N351

Various inhibitors of MIF belonging to the class of compounds having thestructure I(a) were prepared according to the following schemes.

To the suspension of 1M00 (33.0 g; 0.14 mole) in toluene (40 ml) wasadded 108 g of POCl₃ (0.7 mole). The resulting solution was heated underreflux for 1.5 hours. The solvent was distilled under reduced pressureand the residual oil was successively extracted with heptane (control byTLC). Combined heptane fractions were evaporated and the residue washeated with 200 ml of water and filtered off. The yield was 27 g (70%).

After drying at room temperature for 18 hours, the obtained dichlorocompound was transferred to a 250 ml round bottom flask and 150 ml ofacetic acid and 24.0 g of ammonium acetate was added to it. The reactionmixture was heated under reflux for approx. 6 h (control by LCMS andTLC). When no starting material could be detected in the reactionmixture, the hot solution was poured in water and the resultingprecipitate was filtered off. Table 6 provides data on yield (g and %);melting point; mass to charge ratio (M/Z), wherein M/Z=754.1 [3×M]⁺,503.3 [2×M]⁺; τ (8 min. run), and purity as determined by LCMS.

TABLE 6 τ, min Purity, Com- Yield, Yield, m.p., (8 min. % pound g % ° C.M/Z run) (LCMS) 1M00 23.0 92 198–200 754.1; 503.3; 2.97 >96 (Cl) 252.2;206.0

To the solution of 1M00(Cl) (3.27 g; 13.0 mmol) in 20 ml NMP was addedsequentially acylpiperazine (2.34 g; 13.0 mmol) and DABCO (1.46 g; 13.0mmol). The reaction mixture was stirred at 100–120° C. for 15 hours. Thereaction was quenched with 20% NH₄Cl solution and the resultedprecipitate was filtered off and washed with water. The product wasdried in a desiccator over P₂O₅ room temperature under reduced pressure.The product was used in the next reaction without any furtherpurification.

A mixture of chloroquinolone 1N00(Cl) (2.9 g; 13.0 mmol), acylpiperazinetrifluoroacetate AV-0020 (4.0 g; 13.0 mmol), and DABCO (2.91 g; 26.0mmol) in 25 ml NMP was stirred at 100° C. overnight. The mixture waspoured into 50 ml of brine, the solid obtained was filtered off, washedwith water and dried in a desiccator over P₂O₅ at room temperature underreduced pressure. The product was used in the next reaction without anyfurther purification.

The yields and additional information for the obtained compounds areprovided in Table 7. For the compound designated 1M1, X is O and R2 isCOOEt. For the compound designated 1M2, X is S and R2 is COOEt. For thecompound designated 1N1, X is O and R2 is NO₂. For the compounddesignated 1N2, X is S and R2 is NO₂.

TABLE 7 Purity, Yield, Yield, m.p. τ, % g % ° C. M/Z min (LCMS) 1M1 4.792 223–226 dec. 396.2; 350.2 2.67 >94 1M2 4.9 92 220–222 366.2; 412.32.84 >94 1N1 4.5 95 266–267 dec. 369.0 2.73 >92 1N2 4.7 95 265 dec.385.2 2.89 >92

To the suspension of NaH (0.04 g; 1.0 mmol) in dry NMP (3 ml) was addedcompound 1M1 (or 1M2) or 1N1 (or 1N2) (0.8 mmol). After evolution of thegas ceased, the bromide (06-20) (1.0 mmol) was added. The reactionmixture was stirred until no traces of starting material could bedetected (control by LCMS). A 10% solution of NH₄Cl (20 ml) was added tothe reaction and the resulting mixture was extracted with DCM. Compounds1M1 and 1M2 were isolated and purified by preparative HPLC (C-18 silicacolumn, 150 mm×41 mm, 40 ml/min, gradient: water-acetonitrile=from 60:40to 5:95, 20 min). Compounds prepared according to this route aredesignated by the superscript “A” following the compound designation inTable 5.

To the solution of compound 1M1 (or 1M2) or 1N1 (or 1N2) (1.0 mmol) indry DMF (5 ml) was added the bromide (06-20) (2.0 mmol) and K₂CO₃ (200mg). Compounds 1M122; 1M222; 1N122; 1N222 were obtained in 1,4-dioxanewith 4.0 mmol cyclopentyl bromide. The reaction mixture was stirred at80–100° C. for 20–40 hours (control by LCMS). The 10% solution of NH₄Cl(20 ml) was added to the reaction and resulted mixture was extractedwith DCM. Compounds 1M106–1N220 were isolated and purified bypreparative HPLC (C-18 silica column, 150 mm×41 mm, 40 ml/min, gradient:water-acetonitrile=from 80:20 to 5:95, 40 min). Table 5 provides puritydata and other data for the resulting compounds. The compound 1N119*HClwas purified by preparative HPLC (C-18 silica column, 150 mm×41 mm, 40ml/min, gradient: water-acetonitrile-HCl (0.001%)=from 80:20 to 5: 95,40 min). Compounds prepared according to this route are designated bythe superscript “B” following the compound designation in Table 8.Physical properties of the compounds are provided in Table 8.Designations including “(1)” indicate that the compound is a regioisomer.

TABLE 8 τ, min UV Purity, Yield, Yield, m.p. (Wave 254 nm, % Compoundsmg % ° C. M/Z run 10 min.) (LCMS) 1M112^(A) 78 19   151–152.5 506.2;460.4 6.46 >97 1M115^(A) 96 23 177.5–179   516.4; 470.5 5.09 >971N110^(A) 99 29 163–166 423.2 4.96 >99 1M214^(A) 116 29 180.5–182.5508.3; 462.1 5.26 >99 1M106^(A) 98 27 141–143 452.3; 406.3 5.13 >971N106^(A) 152 45 114–116 425.1 5.13 >99 1N114^(A) 97 26 216–217 465.45.08 >96 1N206^(A) 148 42 72–74 441.6 5.41 >97 1N111(1)^(B) 137 29 99–100 465.4; 447.4 5.91 >99 1N115^(B) 157 32 105–110 489.4 5.14  >92¹1N116^(A) 143 34 205–208 527.3 5.69 >99 1N211(1)^(A) 107 22 83–85 481.36.21 >98 1N212^(A) 96 24 73–75 495.5 6.78 >97 1N215^(A) 87 22 220–221505.3 5.38  >93² 1N216^(B) 207 38 228–230 543.2 5.90  >94³ 1N217^(B) 10721 251–252 503.3 5.23  >94⁴ 1N218^(B) 207 39 95–97 525.5 5.92 >961M118^(B) 104 19 159–161 536.4; 490.3 5.64 >97 1M215^(B) 107 20 87–88532.3; 486.3 5.34 >99 1M218^(B) 147 27 110–112 552.4; 506.3 5.89 >991N120^(B) 92 19 179–181 473.4; 455.3 5.49 >98 1N210^(B) 99 23 187–190439.4 5.22 >96 1M107^(B) 138 30 70–72 465.5; 420.3 5.31 >95 1M108^(B)213 45 71–73 478.3; 432.2 4.79 >96 1M110^(B) 142 32 161–162  450.3;404.3; 4.84 >97 350.1 1M111(1)^(B) 185 38 174–175 492.4; 446.2 5.84 >931M207^(B) 131 27 65–67 482.4; 436.4 3.88 >99 1M208B 189 38 71–72 494.5;448.2 5.05 >97 1M211(1)^(B) 148 29 161–163 462.3; 508.5 6.19 >991M212^(B) 105 20 163–165 522.7; 476.3 6.74 >98 1N107^(B) 114 35 103–107439.5 5.40 >99 1N108^(B) 301 59 200–203 451.2 4.83 >97 1N207^(B) 187 2770–72 455.2 5.69 >98 1N214^(B) 146 38 172–175 481.1 5.31 >97 1M113^(B)152 32 147–150 476.3; 430.2 4.71 >99 1M114^(B) 202 41 170–172 492.4;446.2 4.97 >99 1M116^(B) 227 41 185–187 554.4; 508.4 5.67 >98 1M117^(B)147 29 96–98 514.5; 468.6 4.92 >98 1M119^(A) 107 27 65–67  487.4; 441.6;2.95 >96 413.3 1M120^(B) 137 27 165.5–167   500.5; 454.2 5.46 >991M206^(B) 167 36 157–158 468.6; 422.3 5.31 >98 1M210^(B) 107 23 157–158466.3; 420.2 5.13 >99 1M213^(B) 107 42 60–63 492.4; 446.3 5.01 >981M216^(B) 157 28 177–179 570.3; 524.5 5.87 >96 1M217^(B) 102 19 134–135530.4; 484.3 5.18 >97 1M219^(A) 192 48 74–76 503.4; 457.3 3.14 >971M220^(B) 92 18 143–145 516.4; 470.5 5.69 >98 1N112^(A) 182 48 65–68479.2 6.52  >94⁵ 1N113 82 18 95–97 449.1 4.83 >97 1N117^(B) 124 25233.5–235   487.2 4.99 >98 1N118^(B) 107 21   163–163.5 509.5 5.73 >991N119*HCl^(A) 27 5 251–252 460.3 3.10 >98 1N208^(B) 179 38 204–205 467.55.11 >96 1N219^(A) 107 28 258.5–260.5 476.3 3.14 >94 1N220^(B) 202 41231.5–232.5 489.3; 471.5 5.75 >98 1M122 107 23 157–158  464.4; 418.4;5.28  >98⁶ 350.2 1N122 207 47 210–211  437.4; 5.38  >98⁷ 1M222 129 27166–167  480,3; 434.4; 5.56  >99⁸ 366.2 1N222 103 23 110–112 453.2 5.67 >96⁹ ¹HPLC > 96% ²HPLC UV–254 > 94% ³HPLC > 97% ⁴HPLC > 96% ⁵HPLC(UV254) pure > 95%. ⁶HPLC = 100% ⁷HPLC > 94% ⁸HPLC = 100% ⁹HPLC > 96

To a solution of the quinolone 1N01 (or 1M01) (14.08 g; 63.94 mmol) andtriethylbenzylammonium chloride (58.4 g; 256.5 mmol) in MeCN (235 ml)was added 26 ml of POCl₃ (282.4 mmol). The mixture was stirred overnightat room temperature The solvent was removed under reduced pressure andthe residue was stirred in water (335 ml) for 2 hours. The precipitatewas filtered off, washed with water, dried, washed with hot cyclohexane,and dried. Physical properties of the compounds prepared are provided inTable 9.

TABLE 9 Purity, Yield, Yield, m.p., τ, % g % ° C. M/Z min (LCMS)1N01(C1) 5.59 89 258–259 239; 193 3.34 >95 1M01(C1) 8.63 51 95.5–98  266.1; 220.1 3.34 >98

To a solution of 1N01(Cl) (640 mg; 2.68 mmol) in 3 ml DMF was addedsequentially t-butyloxycarbonylpiperazine (500 mg; 2.68 mmol) and DABCO(300 mg; 2.68 mmol). The reaction mixture was stirred at roomtemperature overnight. (For 1M01(Cl), the reaction was stirred at 60° C.overnight). The reaction was quenched with water (15 ml) and theresulted precipitate was filtered off and washed with water. (For 1M01(Cl), the reaction was quenched with 20% NH₄Cl solution (15 ml),extracted with DCM (3×3 ml), dried over Na₂SO₄, the solvent removedunder reduced pressure, and the residue was triturated with hexane. Theprecipitate obtained was filtered off and washed with hexane). Theproduct was dried in a desiccator over P₂O₅ at room temperature underreduced pressure. It was dissolved in 1 ml TFA and kept for 1 hour. Thesolution was triturated with 20 ml of ether, the precipitate wasfiltered off, washed with ether and dried in the air. Physicalproperties of the compounds prepared are provided in Table 10.

TABLE 10 Purity, Yield, Yield, m.p., τ, % g % ° C. M/Z min (LCMS)1MP1TFA 0.84 59 214–215 316.1; 270.1 1.98 >97 dec. 1NP1TFA 1.01 75234–234 589.2; 241.2 1.98 >98 dec.

To a solution of 1N01(Cl) (50 mg; 0.419 mmol) in 3 ml DMF was addedsequentially 3-thienoylpiperazine trifluoroacetate (69 mg; 0.440 mmol)and DABCO (47 mg; 0.419 mmol). The reaction mixture was stirred at roomtemperature overnight. The reaction was quenched with water (15 ml) andthe resulted precipitate was filtered off and washed with water. Theproduct was dried in a desiccator over P₂O₅ at room temperature underreduced pressure. Products prepared according to this scheme aredesignated in Table 11 by the superscript “A” following the compounddesignation.

A mixture of pyrrole-2-carboxylic acid (91 mg; 0.82 mmol) and CDI (133mg; 0.82 mmol) in 2 ml DMF was stirred overnight at room temperature.(In the case of 1M081, 1M091, 1M221, 1M271, 1N081, 1N211—in NMP (1 ml);1M071, 1M51, 1M181, 1M191, 1M211, 1N071, 1N151, 1N181, 1N271—in DMSO (1ml)). Then 1NP1TFA was added and the mixture was stirred at 60° C. for 6hours. (In case of 1M181, 1N181—at room temperature). The mixture wasdiluted with brine 5 ml and extracted with CH₂Cl₂ (3×2 ml). The combinedextracts were washed with water (1 ml), dried over Na₂SO₄ and evaporatedunder reduced pressure. (In the case of 1M071, 1M151, 1M191, 1M211,1N071, 1N151, 1N191, 1N211, the mixture was diluted with water, thepowder precipitated was filtered off, washed with water, dried on theair and dissolved in 5–6N solution of HCl in i-PrOH. The solution washeated under reflux for 10 min. The solvent was evaporated under reducedpressure, the residue was triturated with ether or acetone. Theprecipitate obtained was filtered off to give hydrochloride of thetarget compound). The residues obtained were target products. Productsprepared according to this scheme are designated in Table 11 by thesuperscript “B” following the compound designation. Yield data andproperties of compounds prepared according to Schemes 16 and 17 areprovided in Table 11. Designations including “(2)” indicate that thecompound is a regio isomer.

TABLE 11 Purity, Yield, Yield, m.p. τ, min % Compound mg % ° C. M/Z run10 min.) (LCMS) 1M311^(A) 82 53 113–115 406.3; 361.4 2.38¹ >97 1N111^(A)62 77 182 383.1 3.01¹ >99 1N131^(A) 68 81 252–254 399.0 3.01¹ 1001N311^(A) 138 87 212–214 379.1 2.38¹ >97 1M101^(B) 146 77 225.5–227  409.2; 270.3 4.11 >97 1N121^(A) 87 82 227–230 428.2 4.57 >94 1N161^(B)88 93 157.5–160   383.2 3.46 >99 1N201^(B) 162 84 215–217 461.2 4.81 >971N301^(B) 121 100 227–230 430.3 5.08 >97 1N341^(B) 87 85 225–227 413.04.56  >93² 1N171^(B) 122 41 175–177 398.0 4.36 >95 1N221^(B) 238 81187–188 394.1; 348.2 3.69 >98 1N241^(B) 175 60 258–260 394.1 2.89 >961N351^(B) 210 74 229–231 383.0; 337.3 2.72 >97 1M111(2)^(B) 174 91155.5–158   410.2; 364.2 4.01 >97 1M131^(B) 186 94 137.5–140   426.1;380.1 4.22 >97 1M161^(B) 170 89 189–190  410.1; 364.2; 3.43 >96 346.11M201^(B) 221 97 176–178  490.1; 442.4; 4.67 >95 416.0 1M291^(B) 212 97210–211 471.3; 425.2 4.22 >98 1N091^(B) 227 79 84–87 387.3; 369.23.70 >98 1N141^(B) 124 36   223–224.5 467.3 5.41 >98 1M121^(B) 146 6952–54  455.3; 409.1; 4.45  >94³ 381.2 1M241^(B) 116 59 209–210 421.3;375.1 3.01 >95 1M281 × 1.5 222 98 164–166 459.4; 413.1 4.86 >97 H₂O^(B)1M301^(B) 168 79 67–70  457.3; 411.0; 4.94  >94⁴ 383.1 1M351^(B) 119 62206–208  409.9; 364.3; 2.76 >98 336.4 1N101^(B) 228 80 218–221 382.2;289.2 4.19 >98 1N191*HCl^(B) 250 77 270–273 400.1 2.74 >96 1N231^(B) 25687 217–219 394.1 2.99 >95 1N281^(B) 249 77 283–285 432.2 4.99 >991N291^(B) 254 77 293.5–294   444.4 4.30 >97 1N321^(A) 392 84 146.5–149  369.0 2.85 >99 1N331^(A) 455 94 189–190 385.1 2.99 >99 1M141^(B) 103 45130–131 494.5; 448.2 5.31 >97 1M171^(B) 129 65 125–127  425.0; 379.2;4.22 >96 351.4 1M231^(B) 149 76 49–52 375.1; 421.1 3.01 >96 1M321^(A)247 55 130.5–131.5  396.3; 350.2; 3.04 >98 269.3 1M331^(A) 276 59  113–114.5 412.3; 366.2 2.91 >99 1M341^(B) 114 56 225–227  440.5;394.1; 4.39 >99 270.3 1M071*HCl 233 56 43–45  413.4; 367.1; 2.76 >97270.1 1M081 65 15 143–145 427.2; 270.2 3.09 >99 1M091 171 39 60–62414.4; 270.0 3.59 >99 1M151*HCl 147 27 108–111  431.3; 385.1; 2.18 >99270.3 1M181 124 43 67–69  411.5; 365.3; 3.97    .99 337.4 1M191*HCl 32776 105–107 427.3; 270.3 2.13 >98 1M211(2) 70 14  427.3; 381.4; 2.79 >96270.0 1M221 247 56   160–160.5  421.4; 375.1; 3.60 >93 357.1; 347.31M271 135 31 65–67  422.3; 376.2; 3.56 >94 348.0 1N071*HCl 141 34 83–86386.2 3.14 >96 1N081 85 14 263–265 400.2 3.12 >97 1N151*HCl 227 69198–200 404.2 2.82 >95 1N181 246 86 200–201 384.1 4.00 >97 1N211(2)*HCl71 11 78–80 400.2 2.36 >95 1N271 247 84 214–215  395.1; 349.2; 3.65 >99242.3 ¹Run 8 min. ²HPLC > 98% ³HPLC > 96% ⁴HPLC > 97%

The yield of MIF inhibitors prepared as described in selected schemesabove is provided in Table 12. Designations including “(1)” or “(2)”indicate that the compound is a regio isomer.

TABLE 12 Weight, m.p., No. Compound mg (° C.) 1 1M071*HCl 226 43–45 21M081 58 143–145 3 1M091 164 60–62 4 1M101 139 225.5–227   5 1M106 84141–143 6 1M107 131 70–72 7 1M108 196 71–73 8 1M110 135 161–162 91M111(1) 178 174–175 10 1M111(2) 167 155.5–158   11 1M112 72   151–152.512 1M113 145 147–150 13 1M114 195 170–172 14 1M115 91 177.5–179   151M116 220 185–187 16 1M117 140 96–98 17 1M118 97 159–161 18 1M119 10065–67 19 1M120 130 165.5–167   20 1M121 139 52–54 21 1M122 100 157–15822 1M131 179 137.5–140   23 1M141 96 130–131 24 1M151*HCl 140 108–111 251M161 163 189–190 26 1M171 122 125–127 27 1M181 117 67–69 28 1M191*HCl320 105–107 29 1M201 214 176–178 30 1M206 160 157–158 31 1M207 124 65–6732 1M208 182 71–72 33 1M210 100 157–158 34 1M211(1) 141 161–163 351M211(2)*HCl 112 80–81 36 1M212 98 163–165 37 1M213 200 60–63 38 1M214109 180.5–182.5 39 1M215 100 87–88 40 1M216 150 177–179 41 1M217 95134–135 42 1M218 140 110–112 43 1M219 185 258.5–260.5 44 1M220 85143–145 45 1M221 240   160–160.5 46 1M222 122 166–167 47 1M231 142 49–5248 1M241 109 209–210 49 1M271 128 65–67 50 1M281 215 164–166 51 1M291205 210–211 52 1M301 161 67–70 53 1M311 75 113–115 54 1M321 230130.5–131.5 55 1M331 258   113–114.5 56 1M341 107 225–227 57 1M351 112206–208 58 1N071*HCl 134 83–86 59 1N081 78 263–265 60 1N091 220 84–87 611N101 221 218–221 62 1N106 145 114–116 63 1N107 107 103–107 64 1N108 294200–203 65 1N110 90 163–166 66 1N111(1) 130  99–100 67 1N111(2) 56 18268 1N112 175 65–68 69 1N113 75 95–97 70 1N114 90 216–217 71 1N115 150105–110 72 1N116 133 205–208 73 1N117 117 233.5–235   74 1N118 100  163–163.5 75 1N119*HCl 42 251–252 76 1N120 85 179–181 77 1N121 80227–230 78 1N122 200 210–211 79 1N131 60 252–254 80 1N141 117  223–224.5 81 1N151*HCl 220 198–200 82 1N161 81 157.5–160   83 1N171115 175–177 84 1N181 239 200–201 85 1N191*HCl 243 270–273 86 1N201 155215–217 87 1N206 141 72–74 88 1N207 180 70–72 89 1N208 172 204–205 901N210 92 187–190 91 1N211(1) 100 83–85 92 1N211(2)*HCl 64 78–80 93 1N21289 73–75 94 — — — 95 1N214 139 172–175 96 1N215 80 220–221 97 1N216 200228–230 98 1N217 100 251–252 99 1N218 200 95–97 100 1N219 100258.5–260.5 101 1N220 195 231.5–232.5 102 1N221 231 187–188 103 1N222 96110–112 104 1N231 246 217–219 105 1N241 168 258–260 106 1N271 240214–215 107 1N281 242 283–285 108 1N291 247 293.5–294   109 1N301 114227–230 110 1N311 131 212–214 111 1N321 385 146.5–149   112 1N331 448189–190 113 1N341 80 225–227 114 1N351 202 229–231

Example 15

The following schemes provide a general procedure for synthesizingBoc-derivatives of acids.

A mixture of L-thiazolidine-4-carboxylic acid (1 g; 7.51 mmol, 98%purity, AVOCADO, # 15033), Na₂CO₃ (1.75 g; 16.5 mmol) in H₂O (9 ml) andi-PrOH (1 ml) was stirred until dissolved. Then Boc₂O (1.967 g; 9.01mmol) was added and the mixture was stirred at room temperatureovernight. The suspension obtained was diluted with water (10 ml) andextracted with hexane (5 ml). Lower phase was separated, EtOAc (20 ml)was added and the stirring mixture was acidified to adjust pH 2–3. TheEtOAc phase was separated, water phase was extracted with EtOAc (3×10ml). The combined extracts were washed with water (10 ml), dried overNa₂SO₄ and evaporated under reduced pressure. The residue wascrystallized from ether and the precipitate obtained was filtered off togive after vacuum drying, N-Boc-thiazolidine-4-carboxylic acid (1.03 g;59%).

Example 16

Alkylpiperazines may be synthesized according to the following schemes.

A solution of freshly distilled thionyl chloride (3.9 ml; 0.053 mol) inmethylene dichloride (5 ml) was added dropwise to a stirreed solution of2-thiophenemethanol (4.2 ml; 0.044 mol) and triethylamine (7.4 ml; 0.05mol) in methylene dichloride (25 ml); the temperature being kept below20° C. It was then raised to 40° C. during 1h, poured onto crushed ice,the CH₂Cl₂—phase was separated and dried over MgSO₄. Then it was addeddropwise to a stirred solution of N-Boc-piperazine (2 g; 0.011 mol) andtriethylamine (1.5 ml; 0.011 mol) in CH₂Cl₂ (45 ml). See Nicholas A.Meanwell, Piyasena Hewawasam, Jeanine A. Thomas, J. J. Kim Wright, JohnW. Russel, Marianne Gamberdella, Harold J. Goldenberg, Steven M. Seiler,and George B. Zavoico, Inhibitors of Blood Platelet cAMPPhosphodiesterase. 4. Structural Variation of the Side-Chain Terminus ofWater-Soluble 1,3-Dihydro-2H-imidazo[4,5-b]quinolin-2-one Derivatives,J. Med. Chem. (1993) Vol. 36., pp. 3251–3264; Elena Carceller, ManuelMerlos, Marta Giral, Carmen Almansa, Javier Bartroli, JulianGarcia-Rafanell, and Javier Form, Synthesis and Structure-ActivityRelationships of 1-Acyl-4-((2-methyl-3-pyridyl)cyanomethyl)piperazinesas PAF Antagonists, J. Med. Chem. (1993) No. 36, pp. 2984–2997. Themixture was stirred overnight at room temperature, the solvent wasremoved under reduced pressure, and the residue was extracted withether. The ether solution was evaporated under reduced pressure, theresidue was dissolved in TFA (3.3 ml; 0.043 mol) and kept during 30 min.TFA was removed under reduced pressure, the residue was triturated withether, the precipitate was filtered off and dried on the air to give1-(2-thienylmethyl)piperazine ditrifluoroacetate (3.16 g; 72%). SeeWilliam J. Archer, Robert Cook, and Roger Taylor, Electrophilic AromaticSubstitution. Part 34. Partial Rate Factors for Detritiation ofDithieno[1,2-b:4,3-b′]benzene, Dithieno[1,2-b:3,4-b′]benzene, andDithieno[2,1-b:3,4-b′]benzene, J. Chem. Soc. Perkin Trans. II. (1983)pp. 813–819.

A solution of freshly distilled thionyl chloride (3.9 ml; 0.053 mol) inmethylene dichloride (5 ml) was added dropwise to a stirred solution offurfuryl alcohol (3.8 ml; 0.044 mol) and triethylamine (7.4 ml; 0.05mol) in methylene dichloride (25 ml); the temperature being kept below20° C. The mixture was stirred for 1 h. Then the solvent was evaporated,the residue was dissolved in CH₂Cl₂ (150 ml). The solution obtained wasadded dropwise to a stirred solution of N-Boc-piperazine (2 g; 0.011mol) and triethylamine (4 ml; 0.029 mol) in CH₂Cl₂ (45 ml). The mixturewas stirred overnight at room temperature, the solvent was removed underreduced pressure, and the residue was extracted with ether. The ethersolution was evaporated under reduced pressure, the residue wasdissolved in TFA (3.3 ml; 0.043 mol) and maintained for 30 min. TFA wasremoved under reduced pressure, the residue was titrated with ether, andthe black precipitate obtained was filtered off. Then, the precipitatewas dissolved in 200 ml of MeOH, activated charcoal was added, and themixture was heated under reflux for 30 min. Charcoal was filtered off,the solvent was evaporated, the residue was triturated with ether. Thewhite precipitate obtained was filtered off and dried on the air to give1-(2-furylmethyl)piperazine ditrifluoroacetate (1.64 g; 40%). See R.Lukes and V. Dienstbierova, Synthese von α-methylfural, CollectionCzechoslov. Chem. Commun. (1954) Vol. 19, pp. 609–610.

Example 17

Sulfonamides may be synthesized according to the following schemes.

4-Hydroxy-1-methyl-3-nitro-1H-quinolin-2-one (referred to as 595-01)

A solution of ethylnitroacetate (15.96 g, 120 mmol) was added slowly ina suspension of sodium hydride (60% in mineral oil, 5.28 g, 132 mmol) indimethylacetamide under N₂ atmosphere. The mixture was allowed to stirat room temperature until the evolution of hydrogen gas ceased, thenheated to 90° C. for 30 min. and cooled to room temperature. A solutionof N-methylisatoic anhydride (23.38 g, 132 mmol) in dimethylacetamidewas added slowly and heated overnight at 120° C. The mixture was cooledto room temperature, poured into ice water, and acidified by cold 10%HCl. The solids formed were filtered and washed several times by waterto yield 7.1 g (27%) of yellow solids. Mp 193° C. ¹H NMR (DMSO-d₆):δ3.60 (s, 3H), 7.37 (t, J=7.6 Hz, 1H), 7.58 (d, J=8.5 Hz, 1H), 7.77 (t,J=7.5 Hz, 1H), 8.12 (d, J=7.9 Hz, 1H). EIMS m/z 221 (M+1), 243 (M+23).Anal. (C₁₀H₈N₂O₄) C, H, N.

4-Chloro-1-methyl-3-nitro-1H-quinolin-2-one (Referred to as 595-02)

A suspension of 595-01 (6.2 g, 28.18 mmol) in 70 ml phosphorusoxychloride was heated at 90° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by sodium bicarbonte. The solids formed were filtered anddried to get 4.91 g (73%) of brown solids. Mp 235° C. ¹H NMR (DMSO-d₆):δ3.72 (s, 3H), 7.56 (t, J=7.5 Hz, 1H), 7.78 (d, J=8.6 Hz, 1H), 7.92 (t,J=8.6 Hz, 1H), 8.12 (d, J=7.9 Hz, 1H). EIMS m/z 239 (M+1), 261 (M+23).Anal. (C₁₀H₇N₂O₃Cl) C, H, N.

4-(Thiophene-2-carbonyl)-piperazine-1-carboxylic acid tert-butyl ester(Referred to as 595-03)

2-Thiophenecarbonylchloride (2.04 g, 1.49 mL) was added to a solution oftert-butyl-1-piperazinecarboxylate (2.5 g, 13.4 mmol) and DMAP (20 mg)in pyridine (15 mL) at 0° C. under N₂ atmosphere and stirred at roomtemperature for overnight. The mixture was poured into ice water, theprecipitate was filtered, washed several times with water, and dried toyield white solids (3.5 g, 88%). Mp 76° C. ¹H NMR (DMSO-d₆): δ 1.42 (s,12H), 3.40 (m, 4H), 3.61 (m, 4H), 7.12 (m, 1H), 7.43 (d, J=4.1 Hz, 1H),7.77 (d, J=4.8 Hz, 1H). EIMS m/z 297 (M+1), 319 (M+23). Anal.(C₁₄H₂₀N₂O₃S)C, H, N.

Piperazine-1-yl-thiophen-2-yl-methanone (Referred to as 595-04)

To a solution of 595-03 (3.5 g, 11.8 mmol) in dichloromethane (50 mL)was added trifluoroacetic acid (10 mL). The solution was stirred at roomtemperature for 3 h. The solvent was evaporated under vacuum and theresidue was dissolved in chloroform. The organic phase was washed bysaturated solution of sodium bicarbonate, dried over Na₂SO₄ andevaporated to get 2.20 g (94%) of brown viscous oil. ¹H NMR (DMSO-d₆): δ2.78 (m, 4H), 3.59 (m, 4H), 7.12 (t, J=4.1, 1H), 7.38 (d, J=4.1 Hz, 1H),7.74 (d, J=4.8 Hz, 1H). EIMS m/z 197 (M+1).

1-Methyl-3-nitro-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1H-quinolin-2-one(Referred to as 595-06)

595-04 (1 g, 5.5 mmol) and diisopropylethylamine (1.74 mL, 10 mmol) wasadded to a solution of 595-02 (1.2 g, 5 mmol) in toluene (100 mL) andheated at 100° C. for 15 h. The solvent was removed under vacuum. Thepurification of residue by flash chromatography (CH₂Cl₂/MeOH, 49:1)afforded 1.05 g (53%) yellow solids. Mp 105° C. ¹H NMR (DMSO-d₆): δ 3.19(m, 4H), 3.65 (s, 3H), 3.90 (m, 4H), 7.14 (t, J=4.5 Hz, 1H), 7.43 (t,J=7.5 Hz, 1H), 7.48 (d, J=4.1 Hz, 1H), 7.67 (d, J=8.5 Hz, 1H), 7.80 (m,2H), 8.05 (d, J=8.5 Hz, 1H). EIMS m/z 399 (M+1), 421 (M+23). Anal.(C₁₉H₁₈N₄O₄S)C, H, N.

3-Amino-1-methyl-4-[4-thiophene-2-carbonyl)-piperazine-1-yl]-1H-quinolon-2-one(Referred to as 595-09)

To a suspension of 595-06 (600 mg, 1.5 mmol) in ethanol was added Pd/C(10%, 75 mg). The suspension was stirred under H₂ atmoshphere at 60° C.for 4 h. The hot mixture was filtered through Celite and the filtratewas evaporated to dryness. The residue was recrystallised by ethanol toyield 490 mg (88%) of white solids. Mp 202° C. ¹H NMR (CDCl₃): δ 3.20(br, 2H), 3.49 (br, 2H), 3.65 (br, 2H), 3.79 (s, 3H), 4.13 (br, 2H),4.71 (br, 2H), 7.08 (t, J=4.3 Hz, 1H), 7.22–7.26 (m, 3H), 7.34–7.37 (m,3H), 7.48 (d, J=4.9 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H). EIMS m/z 369 (M+1),391 (M+23). Anal. (C₁₉H₂₀N₄O₂S)C, H, N.

N-{1-Methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-yl}-methanesulfonamide(Referred to as 595-15)

Methanesulfonyl chloride (0.1 mL, 1.3 mmol) was added dropwise to asolution of 595-09 (120 mg, 0.32 mmol) in pyridine (2 mL) under N₂atmosphere and further stirred at room temperature overnight. Thesolvent was evaporated under vacuum and the residue was dissolved inethylacetate. The organic phase was washed successively by saturatedNaHCO₃ solution, water and brine. The organic phase was dried overNa₂SO₄ and evaporated to a residue which was washed by ether to yield103 mg (73%) of white solids. Mp 223° C. ¹H NMR (DMSO-d₆): δ 3.08 (s,3H), 3.31 (m, 4H), 3.64 (s, 3H), 3.95 (m, 4H), 7.15 (t, J=4.0 Hz, 1H),7.33 (t, J=7.6 Hz, 1H), 7.44 (d, J=4.0 Hz, 1H), 7.56 (d, J=8.5 Hz, 1H),7.66 (t, J=7.4 Hz, 1H), 7.79 (d, J=4.9 Hz, 1H), 7.98 (d, J=8.2 Hz, 1H),8.84 (s, 1H). EIMS m/z 447 (M+1), 469 (M+23). Anal. (C₂₀H₂₂N₄O₄S₂) C, H,N.

Other sulfonamides may be prepared according to similar syntheticroutes.

Example 18

Sulfonyls may be synthesized according to the following schemes.

p-Tolylsulfanyl-acetic acid ethyl ester (Referred to as 595-29)

A solution of 4-methylbenzenethiol (5 g, 40.25) in dry THF was addeddropwise to a suspension of NaH (60% in mineral oil, 1.98 g, 48.30) inTHF at room temperature and stirred for 30 min. under N₂ atmosphere.Ethylbromoacetate (4.9 mL, 44.27) was added slowly to this solution andfurther stirred at room temperature for 3 h. The solvent was removedunder vacuum. The residue was dissolved in dil. HCl and extracted byethylacetate. The combined organic phase was washed successively withsaturated NaHCO₃ solution, water and brine then dried over Na₂SO₄.Evaporation of organic phase yielded 8.46 g (99%) colorless oil. ¹H NMR(CDCl₃): δ 1.22 (t, J=7.2 Hz, 3H), 2.32 (s, 3H), 3.57 (s, 2H), 4.14 (q,J=7.2 Hz, 2H), 7.11 (d, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H). EIMS m/z210 (M+1), 233 (M+23).

(Toluene-4-sulfonyl)-acetic acid ethyl ester (Referred to as 595-35)

To a solution of 595-29 (10 g, 47.55 mmol) in dichloromethane was addedm-chloroperbenzoic acid (21.31 g, 95.10 mmol) in portion at 0° C. Themixture was warmed to room temperature and stirred overnight. The solidsformed were filtered and the filtrate was washed successively by 1NNaOH, water and brine. The organic phase was dried over Na₂SO₄ andevaporated to yield 9.8 g (85%) of colorless oil. ¹H NMR (CDCl₃): δ 1.22(t, J=7.2 Hz, 3H), 2.45 (s, 3H), 4.08 (s, 7.2 Hz, 2H), 7.37 (d, J=8.0Hz, 2H), 7.83 (d, J=8.0 Hz, 2H). EIMS m/z243 (M+1), 265 (M+23).

4-Hydroxy-1-methyl-3-(toluene-4-sulfonyl)-1H-quinolin-2-one (Referred toas 595-36)

A solution of 595-35 (9.8 g, 40.49 mmol) was added slowly in asuspension of sodium hydride (60% in mineral oil, 1.78 g, 44.52 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then heated to90° C. for 30 min. and cooled to room temperature. A solution ofN-methylisatoic anhydride (7.88 g, 44.52 mmol) in dimethylacetamide wasadded slowly and heated overnight at 120° C. The mixture was cooled toroom temperature, poured into ice water and acedified by cold 10% HCl.The solids formed were filtered and washed several times by water toyield 5.7 g (43%) of white solids. Mp 191° C. ¹H NMR (DMSO-d₆): δ 2.39(s, 3H), 3.44 (s, 3H), 7.36 (t, J=7.5 Hz, 1H), 7.42 (d, J=8.2 Hz, 2H),7.55 (d, J=8.5 Hz, 1H), 7.81 (t, J=7.1 Hz, 1H), 7.95 (d, J=8.2 Hz, 2H),8.11 (d, J=8.0 Hz, 1H). EIMS m/z 330(M+1), 352 (M+23). Anal.(C₁₇H₁₅NO₄S)C, H, N.

4-Chloro-1-methyl-3-(tolune-4-sulfonyl)-1H-quinolin-2-one (Referred toas 595-46)

A suspension of 595-36 (5.2 g, 5.9 mmol) in 30 mL phosphorus oxychloridewas heated at 130° C. for 30 h. The solvent was evaporated under reducedpressure. The residue was suspended in ice water and neutralized bysodium bicarbonte. The solids formed were filtered and dried to yield2.3 g (43%) of white solids. Mp 193° C. ¹H NMR (DMSO-d₆): δ 2.38 (s,3H), 3.52 (s, 3H), 7.39(d, J=8.0 Hz, 2H), 7.48 (t, J=7.5 Hz, 1H), 7.64(d, J=8.4 Hz, 1H), 7.84 (t, J=7.1 Hz, 1H), 7.94(d, J=8.2 Hz, 2H), 8.32(d, J=8.0 Hz, 1H). EIMS m/z 348 (M+1), 370 (M+23). Anal. (C₁₇H₁₄NO₃SCl)C, H, N.

1-Methyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-3-(tolune-4-sulfonyl)-1H-quinolin-2-one(Referred to as 595-48)

Diisopropylethylamine (0.38 mL, 2.22 mmol) was added to a solution of595-46 (289 mg, 0.83 mmol) and 595-04 (195 mg, 0.99 mmol) in toluene andheated overnight at 105° C. The solution was cooled and the solvent wasevaporated under vacuum. Water was added to the oily residue andsonicated. The solids formed were filtered and washed with water andether to yield yellow solids, 360 mg (86%), mp 213° C. ¹H NMR (DMSO-d₆):δ2.37 (s, 3H), 3.37 (s, 3H), 3.65 (m, 4H), 3.94 (m, 4H), 7.16 (t, J=4.3Hz, 1H), 7.32 (d, J=8.0 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H), 7.49 (d, J=4.0Hz, 1H), 7.54 (d, J=8.5 Hz, 1H), 7.74 (d, J=8.0 Hz, 2H), 7.80 (d, J=4.5Hz, 1H), 8.15 (d, J=8.2 Hz, 1H). EIMS m/z 508 (M+1), 530 (M+23). Anal.(C₂₆H₂₅N₃O₄S₂) C, H, N.

4-Hydroxy-3-methanesulfonyl-1-methyl-1H-quinolin-2-one (Referred to as595-05)

A solution of ethylmethanesulfonylacetate (3.78 g, 22.74 mmol) was addedslowly in a suspension of sodium hydride (60% in mineral oil, 1.07 g, 25mmol) in dimethylacetamide under N₂ atmosphere. The mixture was allowedto stir at room temperature until the evolution of hydrogen gas ceased,then heated to 90° C. for 30 min and cooled to room temperature. Asolution of N-methylisatoic anhydride (4.43 g, 25 mmol) indimethylacetamide was added slowly and heated overnight at 120° C. Themixture was cooled to room temperature, poured into ice water andacidified by cold 10% HCl. The solids formed were filtered and washedseveral times by water to yield 2.76 g (48%) of white solids. Mp 170° C.¹H NMR (DMSO-d₆): δ 3.51 (s, 3H), 3.59 (s, 3H), 7.39 (t, J=7.4 Hz, 1H),7.62 (d, J=8.5 Hz, 1H), 7.84 (t, J=7.0 Hz, 1H), 8.09 (d, J=8.0 Hz, 1H).EIMS m/z 254 (M+1), 276 (M+23). Anal. (C₁₁H₁₁NO₄S) C, H, N.

4-Chloro-3-methanesulfonyl-1-methyl-1H-quinolin-2-one (Referred to as595-14)

A suspension of 595-05 (1.5 g, 5.9 mmol) in 30 mL phosphorus oxychloridewas heated at 130° C. for 30 h. The solvent was evaporated under reducedpressure. The residue was suspended in ice water and neutralized bysodium bicarbonate. The solids formed were filtered and dried to yield773 mg (48%) of white solids solids. Mp 221° C.; ¹H NMR (DMSO-d₆): δ3.48 (s, 3H), 3.68 (s, 3H), 7.49 (t, J=7.8 Hz, 1H), 7.72 (d, J=8.5 Hz,1H), 7.89 (t, J=8.6 Hz, 1H), 8.29 (d, J=8.5 Hz, 1H). EIMS m/z 272 (M+1),294 (M+23). Anal. (C₁₁H₁₀ClNO₃S) C, H, N.

3-Methanesulfonyl-1-methyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1H-quinolin-2-one(Referred to a 95-16)

Diisopropylethylamine (0.38 mL, 2.22 mmol) was added to a solution of595-14 (300 mg, 1.11 mmol) and 595-04 (239 mg, 1.21 mmol) in toluene andheated overnight at 105° C. The solution was cooled and the solvent wasevaporated under vacuum. Water was added to the oily residue andsonicated. The solids formed were filtered and washed with water andether to yield yellow solids, 384 mg (81%), mp 224° C. ¹H NMR (DMSO-d₆):δ 3.36 (s, 3H), 3.52 (m, 4H), 3.60 (s, 3H), 3.91 (m, 4H), 7.16 (t, J=3.5Hz, 1H), 7.37 (t, J=7.5 Hz, 1H), 7.47 (d, J=3.5 Hz, 1H), 7.61 (d, J=8.5Hz, 1H), 7.57(t, J=8.1 Hz, 1H), 7.79 (d, J=4.8 Hz, 1H), 8.10 (d, J=8.5Hz, 1H). EIMS m/z 432 (M+1), 454 (M+23). Anal. (C₂₀H₂₁N₃O₄S₂) C, H, N.

Example 19 4-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid ethylester (Referred to as 595-68)

A solution of diethylmalonate (80 g, 0.50 mol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 22 g, 0.55 mol) indimethylacetamide under N₂ atmosphere. The mixture was allowed to stirat room temperature until the evolution of hydrogen gas ceased, thenheated to 90° C. for 30 min. and cooled to room temperature. A solutionof isatoic anhydride (89.72 g, 0.55 mmol) in dimethylacetamide was addedslowly and heated at 120° C. for 15 h. The mixture was cooled to roomtemperature, poured into ice water and acidified by cold 10% HCl. Thesolids formed were filtered and washed several times by water to yield55 g (47%) of white solids. Mp 173° C. ¹H NMR (DMSO-d₆): δ 1.30 (t,J=6.9 Hz, 3H), 4.33 (q, J=6.9 Hz, 2H), 7.18 (t, J=7.5 Hz, 1H), 7.26 (d,J=8.2 Hz, 1H), 7.62 (t, J=7.2 Hz, 1H), 7.93 (d, J=8.0 Hz, 1H) 11.50 (s,1H), 13.5 (s, 1H). EIMS m/z 234 (M+1), 256 (M+23). Anal. (C₁₂H₁₁NO₄) C,H, N.

2,4-Dichloro-quinoline-3-carboxylic acid ethylester (Referred to as595-72)

A suspension of 595-68 (35 g, 150 mmol) in 200 mL phosphorus oxychloridewas heated at reflux for 30 min. The solvent was evaporated underreduced pressure. The residue was suspended in ice water and neutralizedby sodium bicarbonte. The solid formed were filtered and dried to yield39 g (97%) of white solids. Mp 93° C. ¹H NMR (DMSO-d₆): δ 1.37 (t, J=6.9Hz, 3H), 4.49 (q, J=6.9 Hz, 2H), 7.89 (t, J=8.5 Hz, 1H), 8.02 (t, J=7.2Hz, 1H), 8.10 (d, J=8.3 Hz, 1H), 8.28 (d, J=8.0 Hz, 1H); EIMS m/z 270(M+1), 292 (M+23). Anal. (C₁₂H₉Cl₂NO₂) C, H, N.

4-Chloro-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid ethyl ester(Referred to as 595-76)

Ammonium acetate (12.6 g, 164 mmol) was added to a solution of 595-72(40.17 g, 149 mmol) in acetic acid (150 mL). The mixture was heated at140° C. for 4 h. The solution was cooled and poured into ice water. Thesolids formed were filtered, washed by water and dried to yield whitesolids (34 g, 91%), Mp 186° C. ¹H NMR (DMSO-d₆): δ 1.37 (t, J=6.9 Hz,3H), 4.50 (q, J=6.9 Hz, 2H), 7.87 (t, J=7.2 Hz, 1H), 8.01 (t, J=7.0 Hz,1H), 8.08 (d, J=8.4 Hz, 1H), 8.26 (d, J=8.2 Hz, 1H). EIMS m/z 252 (M+1).Anal. (Cl₂H₁₀ClNO₃) C, H, N.

2-Oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Referred to as 595-77)

To a solution of 595-76 (7 g, 27.8 mmol) in dimethylacetamide was added1,4-diazabicyclo[2.2.2]octane (6.23 g, 55.6 mmol) and 595-04 (6 g, 30.6mmol). The solution was heated at 115° C. for 15 h. The reaction mixturewas cooled and poured into the ice water. The solids formed werefiltered, washed with water and dried to yield white solids (7 g, 62%),mp 198° C. ¹H NMR (DMSO-d₆): δ 1.28 (t, J=6.9 Hz, 3H), 3.12 (m, 4H),3.87 (m, 4H), 4.28 (q, J=6.9 Hz, 2H), 7.15 (t, J=4.3 Hz, 1H), 7.23 (t,J=7.5 Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 7.45 (d, J=3.1 Hz, 1H), 7.54 (t,J=7.4 Hz, 1H), 7.79 (d, J=4.9 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H). EIMS m/z412 (M+1), 434 (M+23). Anal. (C₂₁H₂₁N₃O₄S. 0.5H₂O) C, H, N.

1-(4-Fluorobenzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Referred to as 595-78)

To a suspension of sodium hydride (60% in mineral oil, 0.78 g, 19.46mmol) in DMF was added slowly a solution of 595–77 (7 g, 17.03 mmol) inDMF. The suspension was stirred at room temperature for 30 min.4-Flurobenzylbromide was added to this solution slowly and furtherstirred for 2 h. The mixture was poured into ice water and acidified bycold 10% HCl. The solid formed were separated, washed with water andpurified by flash chromatography (CH₂Cl₂/MeOH, 49:1) to yield 5.9 g(67%) of white solids. Mp 52° C.; ¹H NMR (DMSO-d₆): δ 1.30 (t, J=6.9 Hz,3H), 3.16 (m, 4H), 3.89 (m, 4H), 4.32 (q, J=6.9 Hz, 2H), 7.14–7.17 (m,3H), 7.24–7.27 (m, 2H), 7.31 (t, J=7.6 Hz, 1H), 7.44–7.47 (m, 2H), 7.58(t, J=8.5 Hz, 1H), 7.79 (d, J=4.9 Hz, 1H), 8.02 (d, J=8.5 Hz, 1H). EIMSm/z 520 (M+1), 542 (M+23). Anal. (C₂₈H₂₆FN₃O₄S. H₂O) C, H, N.

Example 20

The following describes the synthesis of a library of compounds ofgeneral structure 1(a) and 1(b) as depicted above. Compounds includingan “M” in the designation incorporate a —COOEt moiety. Compoundsincorporating an “N” in the designation incorporate a —NO₂ moiety. Thetwo digits following “M” or “N” correspond to the numerical designationfor the functional group R2 and R3 respectively, provided below. Thedigit preceeding “M” or “N” corresponds to the numerical designation forthe functional group R1. The compounds have the following structures,except for those with designations including “+i”.

In compounds that have designations including “+i”, R₃ is a substituenton the oxygen atom of the quinolone group rather than the nitrogen atom,i.e., a compound of structure 1(b), as depicted below. The designation“i” appears elsewhere in the preferred embodiments, and refers to asubstituent on the oxygen atom of the quinolone group rather than thenitrogen atom.

Numerical Designations for R1 Functional Groups

Hydrogen Methyl Chlorine 1 2 3

Numerical Designations for R2 Functional Groups

Numerical Designations for R3 Functional Groups

The numerical designations of the MIF inhibitors prepared are providedin Table 13.

TABLE 13 R3 = 2 R3 = 4 R3 = 5 R3 = 6 R3 = 1Methyl

1N11 1N12 1N13 1N14 1N15 1N21 1N22 1N23 1N24 1N25 1N31 1N32 1N33 1N341N35 1N41 1N42 1N43 1N44 1N45 1N51 1N52 1N53 1N54 1N55 + i 1N61 1N621N63 1N64 1N65 2N11 2N12 + i 2N13 2N14 2N15 2N21 2N22 2N23 2N24 2N252N31 2N32 2N33 2N34 2N35 2N41 2N42 2N43 2N44 2N45 2N51 2N52 2N53 2N542N55 + i 2N61 2N62 2N63 2N64 2N65 1M11 1M12 1M13 1M14 1M15 + i 1M21 1M221M23 1M24 1M25 + i 1M31 1M32 1M33 1M34 1M35 + i 1M41 1M42 1M43 1M441M45 + i 1M51 1M52 1M53 1M54 1M55 1M61 1M62 1M63 1M64 1M65 2M11 2M122M13 2M14 2M15 2M21 2M22 2M23 2M24 2M25 + i 2M31 2M32 2M33 2M34 2M35 + i2M41 2M42 2M43 2M44 2M45 + i 2M51 2M52 2M53 2M54 2M55 2M61 2M62 2M632M64 2M65 + i 3M11 3M12 3M13 3M14 3M15 + i 3M21 3M22 3M23 3M24 3M25 3M313M32 3M33 3M34 3M35 + i 3M41 3M42 3M43 3M44 3M45 + i 3M51 3M52 3M53 3M543M55 + i 3M61 3M62 3M63 3M64 3M65 + i

Details of reaction schemes for preparing intermediates or MIFinhibitors are provided below.

All of the following compounds were obtained using a similar or the sameprocedure: Compound 1M21: yield 176 mg, 56.56%; Compound 1M31: yield 64mg, 20.60%; Compound 1M41: yield 110 mg, 35.48%; Compound 1M51: yield139 mg, 44.18%; Compound 1M61: yield 88 mg, 28.37%; Compound 2M13 yield144 mg, 38.09%; Compound 2M21: yield 113 mg, 36.73%; Compound 2M23:yield 137 mg, 36.16%; Compound 2M31: yield 27 mg, 8.67%; Compound 2M33:yield 141 mg, 37.45%; Compound 2M41: yield 72 mg, 23.30%; Compound 2M43:yield 117 mg, 31.54%; Compound 2M51: yield 65 mg, 21.20%; Compound 2M53yield 91 mg, 24.87%; Compound 2M61: yield 113 mg, 36.94%; Compound 2M63:yield 127 mg, 33.99%; Compound 3M11: yield 58 mg, 19.00%; Compound 3M21:yield 134 mg, 43.32%; Compound 3M31: yield 117 mg, 37.50%; Compound3M41: yield 141 mg, 45.83%; Compound 3M51 yield 119 mg, 38.42%; Compound3M61: yield 142 mg, 45.85% and Compound 3M63: yield 40 mg, 11.00%.

Example 21

Boc-derivatives of acids were prepared according to the followingreaction scheme.

Yield and purity for compounds prepared according to Scheme 25 areprovided in Table 13.

TABLE 13 AV-0010 Yield, g Yield, % Purity, % CMS AV-0020 25.0 55 >90AV-0030 42.0 52 >90 AV-0040 39.0 79 >90 AV-0050 49.0 86 >90 AV-0060 36.082 >90 AV-0070 43.0 88 >90

Example 22

A series of MIF inhibitors was prepared according to the followingreaction schemes. The reactants are abbreviated as follows:DCM=Dichloromethane; DMA=Dimethylacetamide; DMF═N-Dimethylformamide;HOAc=Acetic acid; MeCN=Acetonitrile; DABCO=Triethylenediamine;TEBAC=Benzyltriethylammonium chloride; NMP=1-Methyl-2-pyrrolidinone;BOC=tert-BuOCO; PPA=Polyphosphoric acid; TFA=Trifluoroacetic acid.

To a intensively stirred solution of 2-amino-5-methylbenzoic acid (67.7g, 0.45 mole) in a mixture of 250 ml of dioxane and 150 ml of toluene, asolution of diphosgene (97.6 g, 0.49 mole) in 80 ml of dioxane was addeddropwise. The reaction mixture was stirred for 12 hours and after thatthe precipitate was filtered off and washed with ether. The filtrate andether fractions were combined and the solvent was removed under reducedpressure. The residue was titrated with hexane and the resultingprecipitate was filtered off, washed with hexane and dried at roomtemperature overnight. Compound 2000: yield 66.5 g (84%), purity 93%(LCMS).

To a stirred suspension of NaH (18.9 g, 0.47 mole) in dry DMA, a malonicester was added dropwise. The reaction mixture was stirred for 20 minand cooled to 30° C. The isatoic anhydride was added portion-wise to theresulting solution. The reaction mixture was heated at 130–150° C. for10 hours and after that the DMA was distilled off. The residue wastitrated with water and acidified using 10% HCl to pH=3. The resultingprecipitate was filtered off and washed with water. The solid materialwas placed in a 2 L conical flask, 1 L of water was added and the pH wasadjusted to 12–13 using K₂CO₃. The resulting solution was filtered andfiltrate was acidified by 10% HCl to pH=2–3. The precipitate wasfiltered off, washed with ether and crystallized from dioxane. Compound2M00: yield 23.3 g (24%), purity; 90% (LCMS).

To the suspension of 2M00 (23.0 g, 0.093 mole) in toluene (40 ml) wasadded 71.3 g of POCl₃ (43 ml, 0.465 mole). The resulting solution washeated under reflux for 1.5 hours. The solvent was distilled underreduced pressure and the residual oil was successively extracted withheptane (control by TLC). Combined heptane fractions were evaporated andthe residue was heated with 200 ml of water and filtered off. Afterdrying at room temperature for 18 hours, the dichloro compound obtainedwas transferred to a 250 ml round bottom flask and 90 ml of acetic acidand 8.0 g of ammonium acetate was added to it. The reaction mixture washeated under reflux for approx. 2 h (control by LCMS and TLC). When nostarting material could be detected in the reaction mixture, the hotsolution was poured in water and the resulting precipitate was filteredoff. Yield and purity for compounds prepared according to the abovescheme are provided in Table 14.

TABLE 14 Compound Yield, g Yield, % Purity, % LCMS AV-1M00(Cl) 50.0070 >90 AV-2M00(Cl) 10.96 44 >90 AV-3M00(Cl) 30.00 70 >90

Method A: To the solution of 2M00 1.0 g (3.77 mmol) in DMA was addedsequentially acylpiperazine 0.75 g (4.16 mmol) and DABCO 0.84 g (7.5mmol). The reaction mixture was stirred at 100–120° C. for 15 hours. Thereaction was quenched with 20% NH₄Cl solution and the resultedprecipitate was filtered off and washed with water. The product wasdried in desiccator over P₂O₅ at room temperature under reducedpressure. The product was used in the next reaction without any furtherpurification.

Method B: A mixture of chloroquinolone 2M00 1.0 g (3.77 mmol),acylpiperazine trifluoroacetate, AV-0050 1.55 g (4.14 mmol) and DABCO0.84 g (7.53 mmol) in DMF 3 ml was stirred at 101° C. overnight themixture was poured in 50 ml of brine, the solid obtained was filteredof, washed with water, and dried in desiccator over P₂O₅ at roomtemperature under reduced pressure. The product was used in the nextreaction without any further purification

The yields of the obtained compounds are provided in Table 15.

TABLE 15 Compound Method Yield, g Yield, % Purity, % CMS 1M10 A 1.3888 >90 1M20 A 1.47 90 >90 1M30 A 1.49 89 >90 1M40 A 1.67 95 >90 1M50 A1.78 94 >90 1M60 A 1.58 91 >90 2M10 A 1.16 75 >90 2M20 A 1.22 76 >902M30 A 1.24 76 >90 2M40 A 1.34 78 >90 2M50 B 1.83 99 >90 2M60 A 1.3178 >90 3M10 A 1.05 70 >90 3M20 A 1.21 78 >90 3M30 A 1.26 79 >90 3M40 A1.41 85 >90 3M50 A 1.64 92 >90 3M60 A 1.28 78 >90

To the suspension of NaH 0.03 g (0.8 mmol) in dry DMF (1 ml) was addedcompound 2M10 0.30 g (0.7 mmol). After evolution of the gas ceased, thebenzylbromide 0.19 g (1.1 mmol) was added. The reaction mixture wasstirred until no traces of starting material could be detected (controlby LCMS). The 20% solution of NH₄Cl (2 ml) was added to the reaction andresulted mixture was extracted with DCM. Compound 2M12 was isolated andpurified by preparative HPLC (C-18 silica column, 150 mm×41 mm, 40ml/min, gradient: water-acetonitrile=from 60:40 to 5:95, 20 min):Compound 2M12: yield 114 mg (31%), purity >99% (HPLC):

Method A: To the suspension of NaH 0.03 g (0.8 mmol) in dry DMF (2 ml)was added compound 1M60 300 mg (0.7 mmol). After evolution of the gasceased (˜30 min) the solution of dimethylaminoethyl chloride (2.1 mmol)in ether was added. The reaction mixture was heated at 100° C. (etherwas removed by distillation) for 12 h. The solution was cooled to roomtemperature and pH of the mixture was adjusted to pH 9 by 1% solution ofAcOH in water. The mixture was extracted with DCM (3×3 ml) and combinedDCM fractions were washed with brine and dried over MgSO₄. DCM wasremoved on a rotary evaporator and the product was purified by prep TLC(AnalTech silica gel GF, 1000 gm, eluent: CHCl₃: EtOH=4:1). Compound1M64: yield 84 mg (24%), purity>99% (HPLC).

Method B: To a suspension of NaH 0.114 g (2.84 mmol; of 60% dispersionin mineral oil) in 3 ml NMP, the quinolone 2M50 0.33 g (0.676 mmol) wasadded portion-wise. After the evolution of the gas ceased (˜30 min) themixture was stirred for 30 min at room temperature anddimethylaminoethylchloride hydrochloride 0.195 g (1.35 mmol) was added.The resulting mixture was heated at 100° C. overnight. The reactionmixture was cooled and poured in water (25 ml) and the solid obtainedwas filtered off, washed with water and dried at 85° C. overnight. Thetarget isomer was isolated by prep. TLC (AnalTech silica gel GF, 1500gm, eluent: 10% of triethylamine in EtOAc, lower spot). Compound 2M54:yield 68 mg (18%).

To the suspension of NaH (0.03 g, 0.8 mmol) in dry DMF (2 ml) was addedcompound 1 M60 (300 mg, 0.7 mmol). After evolution of the gas ceased(˜30 min) the solution of dimethylaminoethyl chloride (2.1 mmol) inether was added. The reaction mixture was heated at 100° C. (ether wasremoved by distillation) for 12 h. The solution was cooled to roomtemperature and the pH of the mixture was adjusted to pH=9 by a 1%solution of AcOH in water. The mixture was extracted with DCM (3×3 ml)and the combined DCM fraction was washed with brine and dried overMgSO₄. DCM was removed on a rotary evaporator and the product waspurified by prep. TLC (AnalTech silica gel GF, 1000 gm, eluent:CHCl₃=EtOH=4:1). Compound 1M64: yield 57 mg, purity >99% (HPLC). All ofthe following compounds were obtained using similar or the sameprocedure: Compound 1M34: yield 77 mg, 22.00%; Compound 1M54: yield 58mg, 16.88%; Compound 1M64: yield 84 mg, 24.00%; Compound 2M14: yield 57mg, 16.25%; Compound 2M34: yield 63 mg, 17.95%; and Compound 2M64: yield42 mg, 12.00%.

Compounds 1M15 . . . 3M65: All compounds listed below were obtainedusing the procedure described above for compound 1M64: Compound 3M15: 36mg; 13%; Compound 3M65: 5 mg; 1%; Compound 1M65: 31 mg; 9%; Compound1M45: 34 mg; 10%; Compound 1M55: 51 mg; 15%; Compound 1M35: 51 mg; 14%;Compound 3M45: 52 mg; 15%; Compound 3M25: 24 mg; 7%; Compound 3M35: 71mg; 20%; Compound 3M35i; 16 mg; 4%; Compound 3M15i; 22 mg; 8%; Compound1M65i: 20 mg; 6%; Compound 1M45i: 27 mg; 8%; Compound 1M35i-28 mg; 8%;Compound 3M65i: 23 mg; 6%.

To the solution of p-toluidine 10 g (93.3 mmol) and Et₃N (13.6 ml) inDCM (100 ml) was added drop-wise monoethylmalonate chloride (17.72 ml)at 0–5° C. (ice-water bath). After the completion of the reaction(control by TLC) the reaction mixture was poured into water (300 ml) andpH was adjusted to 2 by HCl (c.). The organic layer was separated andthe water phase was extracted by DCM (3×50 ml). Combined DCM extractswere washed with brine (50 ml) and dried over sodium sulfate. DCM wasremoved on rotary evaporator and a residue was dissolved in mixture of600 ml of MeOH and 400 ml of 1 N NaOH. The reaction mixture was heatedunder reflux for 3 hours, cooled to room temperature and acidified by 2NHCl to pH 2. MeOH was removed under reduced pressure and water wasextracted by EtOAc (3×100 ml). Organic phase was washed with brine,dried over sodium sulfate and solvent was removed under reducedpressure. To the residue was added 40 g of PPA and the mixture stirredon magnetic stirrer and heated at 170° C. for 3 hours. The reactionmixture was cooled to room temperature and was slowly diluted with 500ml of 1 NHCl. The pH of the resulting solution was adjusted by asolution of 20% NaOH in water to pH 4. Formed precipitate was filteredoff, washed with water and dried in a desiccator over NaOH overnight.The yield of 05 was 13.2 g (81%).

To the solution of 5-methyl-2,4-dihydroxyquinoline 13.2 g in glacialacetic acid (200 ml) was slowly added 25 ml of HNO₃ (63%). The reactionmixture was heated at 90° C. for 30 min, cooled to the room temperatureand poured into water (700 ml). The formed precipitate was filtered offand washed with water. The obtained compound was dried over NaOH indesiccator overnight. The yield of 06 was 7.68 g (44%).

To the stirred suspension of dihydroxyquinolinone 01 (50 g) in glacialacetic acid (600 ml) was added 98 ml of HNO₃ (63%). The reaction mixturewas heated at 90° C. for 30 min and cooled to room temperature. Theformed precipitate was filtered off and washed with water (5×100 ml).The obtained compound was dried over P₂O₅ in a desiccator overnight. Theyield of INOO was 52.7 g (82%).

To a solution of 5-chloroisatoic anhydride 3000 15 g (75.91 mmol) in DMF(75 ml) was added potassium carbonate anhydrous (8.85 g) and methyliodide 14.46 g (114 mmol). The reaction mixture was stirred at roomtemperature for 18 h. and poured into ice water. The precipitate wasfiltered off, washed with water and dried over P₂O₅ in a desiccatorovernight. Compound 3001: yield 15.3 g (95%), purity >90% (LCMS).Compound 3003: yield 18.2 g (79%), purity >90% (LCMS). All of thefollowing compounds were obtained using similar or the same procedure:Compound 3002: 16.1 g, 74% yield, purity>90% (LCMS); Compound 3003: 18.2g, 78.5% yield, purity >90% (LCMS)

To the stirred solution of ethyl nitroacetate 7.5 ml (68 mmol) in 50 mlof DMF was added portion-wise to 2.85 g of NaH. After evolution ofhydrogen ceased, the mixture was heated at 80° C. for 15 min. Thesolution of N-methyl isatoic anhydride 3001 15 g (71 mmol) in 60 ml ofDMF was added over a period of 15 min. after which the reaction washeated at 120° C. for 18 h. The solvent was removed by distillation, theresidue was dissolved in water and acidified with 6 N HCl to pH=4. Theprecipitate was collected, washed with water and dried in a desiccatorover NaOH overnight. Compound 3N01: yield 16.6 g (92%), purity >90%(LCMS). Compound 3N03: yield 18.5 g (89%), purity >90% (LCMS). Compound3N03: yield 18.5 g (89%) purity >90% (LCMS).

To a solution of quinolone 3N00 18.8 g (78.1 mmol) andtriethylbenzylammonium chloride 71 g (312 mmol) in MeOH (290 ml), POCl₃32 ml (344 mmol) was added. The mixture was stirred overnight. Thesolvent was removed under reduced pressure, and the residue was stirredin water (290 ml) for 3 h. The solid precipitated was filtered off,washed with water dried, washed with hot cyclohexane, dried and doublecrystallized from THF-hexane. Compound 3N00 (Cl): yield 5.59 g (28%),purity >95%(LCMS).

A mixture of chloroquinolone 3N00(Cl) 0.30 g (1.16 mmol), acylpiperazinetrifluoroacetate AV-0050 0.45 g (1.22 mmol) and DABCO 0.26 g (2.32 mmol)in DMF 2 ml was stirred overnight. Then the mixture was poured in water(15 ml), the solid obtained was filtered off, washed with water anddried over P₂O₅ in desiccator overnight. The product was used in thenext reaction without any further purification. Compound 3N50: yield0.50 g (90%), purity >95% (LCMS).

The preferred embodiments have been described in connection withspecific embodiments thereof. It will be understood that it is capableof further modification, and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practices in theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as fall within the scopeof the invention and any equivalents thereof. All references citedherein, including but not limited to technical literature references,granted patents, and patent applications are incorporated herein byreference in their entireties.

1. A compound having a structure:

or a stereoisomer or a pharmaceutically acceptable salt thereof,wherein: X is oxygen or sulfur; Y is selected from the group consistingof —NO₂ and —C(═O)OR₅; Z is —CH₂— or —C(═O)—; R₁ is selected from thegroup consisting of C₁₋₁₀ alkyl and aryl C₁₋₁₀ alkyl, wherein R₁ isunsubstituted or substituted with at least one substituent selected fromthe group consisting of halogen, alkoxy, alkylamino, dialkylamino, andketo; R₂ and R₃ are independently selected from the group consisting ofhalogen, hydrogen, and C₁₋₆ alkyl; R₇ is selected from the groupconsisting of cyclopentyl, phenyl, pyrazolyl, thiadiazolyl, isoxazolyl,imidazolyl, pyrrolyl, indolyl, isoquinolinyl, pyridinyl,tetrahydrothiophenyl, thienyl, furyl, tetrahydrofuranyl, thiazolidinyl,pyrazinyl, pyrrolidinyl, and piperidinyl, wherein R₇ is unsubstituted orsubstituted with at least one substituent selected from the groupconsisting of halogen, alkoxy, nitro, and alkylamino; and R₅ is C₁₋₆alkyl.
 2. The compound of claim 1, wherein Z is —CH₂—.
 3. The compoundof claim 2, wherein X is oxygen.
 4. The compound of claim 2, wherein Yis —C(═O)OCH₂CH₃.
 5. The compound of claim 2, wherein Y is —NO₂.
 6. Thecompound of claim 2, wherein R₇ is


7. The compound of claim 2, wherein R₇ is


8. The compound of claim 1, wherein Y is —NO₂.
 9. The compound of claim8, wherein X is oxygen.
 10. The compound of claim 8, wherein R₇ is


11. The compound of claim 8, wherein R₇ is


12. The compound of claim 1, wherein Y is —C(═O)OCH₂CH₃.
 13. Thecompound of claim 12, wherein X is oxygen.
 14. The compound of claim 12,wherein R₇ is


15. The compound of claim 12, wherein R₇ is


16. The compound of claim 1, wherein R₇ is


17. The compound of claim 16, wherein X is oxygen.
 18. The compound ofclaim 1, wherein R₇ is


19. The compound of claim 18, wherein X is oxygen.
 20. The compound ofclaim 1, wherein X is oxygen.
 21. A composition comprising a compound ofclaim 1 in combination with a pharmaceutically acceptable carrier ordiluent.
 22. A compound having a structure:

or a stereoisomer or a pharmaceutically acceptable salt thereof,wherein: X is oxygen or sulfur; Y is selected from the group consistingof —NO₂ and —C(═O)OR₅; Z is —CH₂— or —C(═O)—; R₁ is selected from thegroup consisting of C₁₋₁₀ alkyl and aryl C₁₋₁₀ alkyl, wherein R₁ isunsubstituted or substituted with at least one substituent selected fromthe group consisting of halogen, alkoxy, alkylamino, dialkylamino, andketo; R₂ and R₃ are independently selected from the group consisting ofhalogen, hydrogen, and C₁₋₆ alkyl; R₇ is selected from the groupconsisting of cyclopentyl, phenyl, pyrazolyl, isoxazolyl, imidazolyl,pyrrolyl, indolyl, isoquinolinyl, pyridinyl, tetrahydrothiophenyl,thienyl, furyl, tetrahydrofuranyl, pyrazinyl, pyrrolidinyl, andpiperidinyl, wherein R₇ is unsubstituted or substituted with at leastone substituent selected from the group consisting of halogen, alkoxy,nitro, and alkylamino; and R₅ is C₁₋₆ alkyl.
 23. The compound of claim22, wherein Z is —CH₂—.
 24. The compound of claim 23, wherein X isoxygen.
 25. The compound of claim 23, wherein Y is —C(═O)OCH₂CH₃. 26.The compound of claim 23, wherein Y is —NO₂.
 27. The compound of claim23, wherein R₇ is


28. The compound of claim 23, wherein R₇ is


29. The compound of claim 22; wherein Y is —NO₂.
 30. The compound ofclaim 29, wherein X is oxygen.
 31. The compound of claim 29, wherein R₇is


32. The compound of claim 29, wherein R₇ is


33. The compound of claim 22, wherein Y is —C(═O)OCH₂CH₃.
 34. Thecompound of claim 33, wherein X is oxygen.
 35. The compound of claim 33,wherein R₇ is


36. The compound of claim 33, wherein R₇ is


37. The compound of claim 22, wherein R₇ is


38. The compound of claim 37, wherein X is oxygen.
 39. The compound ofclaim 22, wherein R₇ is


40. The compound of claim 39, wherein X is oxygen.
 41. The compound ofclaim 22, wherein X is oxygen.
 42. A composition comprising a compoundof claim 22 in combination with a pharmaceutically acceptable carrier ordiluent.